Manganese supplementation for control of glycosylation in mammalian cell culture process

ABSTRACT

The present invention pertains to a cell culture medium comprising manganese as a media supplement, which was shown to control recombinant protein glycosylation and methods of using thereof. The present invention further pertains to a method of controlling or manipulating glycosylation of a recombinant protein of interest in a large scale cell culture.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention pertains to a cell culture medium comprisingmanganese as a media supplement that was shown to control recombinantprotein antennarity and/or glycosylation and methods of using thereof.The present invention further pertains to a method of controlling ormanipulating antennarity and/or glycosylation of a recombinant proteinof interest in a large scale cell culture, comprising controlling ormanipulating the concentration of manganese in the cell culture medium.

Background Art

Over the last few decades, much research has focused on the productionof therapeutic recombinant proteins, e.g., monoclonal antibodies. Whilemedia containing sera or hydrolysates has been utilized, chemicallydefined media were also developed in order to eliminate the problematiclot-to-lot variation of complex components (Luo and Chen, Biotechnologyand Bioengineering 97(6):1654-1659 (2007)). An improved understanding ofcell culture has permitted a shift to chemically defined medium withoutcompromising growth, viability, titer, etc. To date optimized chemicallydefined processes have been reported with titers as high as 7.5-10 g/L(Huang et al., Biotechnology Progress 26(5):1400-1410 (2010); Ma et al.,Biotechnology Progress 25(5):1353-1363 (2009); Yu et al., Biotechnologyand Bioengineering 108(5):1078-1088 (2011)). In general, the high titerchemically defined processes are fed batch processes with cultivationtimes of 11-18 days. The process intensification has been achievedwithout compromising product quality while maintaining relatively highviabilities.

Achievement of a robust, scalable production process includes more thanincreasing the product titer while maintaining high product quality. Theprocess must also predictably require the main carbohydrate sourceremain constant, such that the feeding strategy does not need to changeacross scales. As many processes use glucose as the main carbohydrate,and have lactate and ammonium as the main byproducts, the time course ofthese three critical chemicals should also scale.

A number of reports have demonstrated mammalian host cell-specificprocessing of N-glycans associated with recombinant proteins (James etal., Bio/Technology, 13:592-596 (1995); Lifely et al., Glycobiology,5:813-822 (1995)). These differences may be important for therapeuticproteins as they can directly alter the antigenicity, rate of clearancein vivo, and stability of recombinant proteins (Jenkins et al., NatureBiotechnol. 14:975-981 (1996)). Thus, it is important not only to beable to characterize glycans bound to a therapeutic recombinant proteinto predict the consequences for in vivo safety and efficacy, but also tounderstand the cellular controls underpinning glycan processing in apotential host cell enabling the implementation of appropriatestrategies to control cellular glycosylation (Grabenhosrt et al.,Glycoconjug. J., 16:81-97 (1999); James and Baker, Encyclopedia ofbioprocess technology: Fermentation, biocatalysis and bioseparation. NewYork: John Wiley & Sons. p. 1336-1349 (1999)).

Thus, there is a need in the art for identification of methods that canpredictably control glycosylation of proteins of interest.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method forachieving a predetermined glycosylation profile of an anti-α4-integrinantibody comprising providing manganese to a cell culture at aconcentration that falls within a target manganese concentration range,wherein the cell culture comprises host cells producing theanti-α4-integrin antibody.

In another aspect, the invention is directed to supplementing the cellculture with manganese if the manganese concentration in the cellculture is below the target manganese concentration range.

In another aspect, the invention is directed to a method for achieving apredetermined glycosylation profile of an anti-α4-integrin antibodycomprising (i) determining a manganese concentration in a component of acell culture medium, (ii) if the manganese concentration is below atarget manganese concentration range, supplementing the cell culturemedium with the component to achieve a manganese concentration withinthe target manganese concentration range, and (iii) culturing arecombinant host cell producing an anti-α4-integrin antibody in the cellculture medium comprising the cell culture medium component.

In another aspect, the invention is directed to a method for achieving apredetermined glycosylation profile of an anti-α4-integrin antibodycomprising (i) determining a manganese concentration in a component of acell culture medium, (ii) if the manganese concentration is below atarget manganese concentration range, adding manganese to the componentof the cell culture medium to achieve a manganese concentration withinthe target manganese concentration range, (iii) producing a cell culturemedium using the component of cell culture medium with the targetmanganese concentration, and (iv) culturing a recombinant host cellproducing an anti-α4-integrin antibody in the cell culture mediumcomprising the cell culture medium component with the target manganeseconcentration.

In another aspect, the invention is directed to a method for optimizinga cell culture medium for the production of an anti-α4-integrin antibodycomprising (i) determining the amount of manganese in a cell culturemedium or a component used to produce a cell culture medium, and (ii) ifthe amount of manganese is below a target range, supplementing the cellculture medium or the component of the cell culture medium withmanganese to achieve an amount of manganese within the target range,wherein the target range is sufficient to produce anti-α4-integrinantibody with a predetermined glycosylation profile.

In another aspect, the invention is directed to a method for optimizinga cell culture medium for the production of an anti-α4-integrin antibodycomprising (i) determining the amount of manganese in a cell culturemedium or a component used to produce a cell culture medium, and (ii) ifthe amount of manganese is above a target range, removing manganese fromthe cell culture medium or the component of the cell culture medium toachieve an amount of manganese within the target range, wherein thetarget range is sufficient to produce anti-α4-integrin antibody with apredetermined glycosylation profile.

In one embodiment, the predetermined glycosylation profile of theanti-α4-integrin antibody comprises 13 to 32% galactosylation. In afurther embodiment, the predetermined glycosylation profile of theanti-α4-integrin antibody comprises 18 to 31% galactosylation. In afurther embodiment, the predetermined glycosylation profile of theanti-α4-integrin antibody comprises about 24% galactosylation.

In one embodiment, the predetermined glycosylation profile of theanti-α4-integrin antibody comprises 0.7 to 3.6% sialylation. In afurther embodiment, the predetermined glycosylation profile of theanti-α4-integrin antibody comprises 1.0 to 2.2% sialylation. In afurther embodiment, the predetermined glycosylation profile of theanti-α4-integrin antibody comprises about 1.6% sialylation.

In one embodiment, the target manganese concentration range in the cellculture for anti-α4-integrin antibody is 0.025 μM to 10 μM. In anotherembodiment, the target manganese concentration range in the cell culturefor anti-α4-integrin antibody is 0.1 μM to 2.5 μM. In anotherembodiment, the target manganese concentration range in the cell culturefor anti-α4-integrin antibody is 0.2 μM to 2 μM.

In one embodiment, the target manganese concentration range in the cellculture for anti-α4-integrin antibody is at day 0 between 0.002 μM and0.4 μM and at day 13 between 0.04 μM and 1 μM.

In one aspect, the present invention is directed to a method forachieving a predetermined glycosylation profile of an interferon beta-1apolypeptide comprising providing manganese to a cell culture at aconcentration that falls within a target manganese concentration range,wherein the cell culture comprises host cells producing the interferonbeta-1a polypeptide. In another aspect, the invention is directed tosupplementing the cell culture with manganese if the manganeseconcentration in the cell culture is below the target manganeseconcentration range.

In another aspect, the invention is directed to a method for achieving apredetermined glycosylation profile of an interferon beta-1a polypeptidecomprising (i) determining a manganese concentration in a component of acell culture medium, (ii) if the manganese concentration is below atarget manganese concentration range, supplementing the cell culturemedium with the component to achieve a manganese concentration withinthe target manganese concentration range, and (iii) culturing arecombinant host cell producing an interferon beta-1a polypeptide in thecell culture medium comprising the cell culture medium component.

In another aspect, the invention is directed to a method for achieving apredetermined glycosylation profile of an interferon beta-1a polypeptidecomprising (i) determining a manganese concentration in a component of acell culture medium, (ii) if the manganese concentration is below atarget manganese concentration range, adding manganese to the componentof the cell culture medium to achieve a manganese concentration withinthe target manganese concentration range, (iii) producing a cell culturemedium using the component of cell culture medium with the targetmanganese concentration, and (iv) culturing a recombinant host cellproducing an interferon beta-1a polypeptide in the cell culture mediumcomprising the cell culture medium component with the target manganeseconcentration.

In another aspect, the invention is directed to a method for optimizinga cell culture medium for the production of an interferon beta-1apolypeptide comprising (i) determining the amount of manganese in a cellculture medium or a component used to produce a cell culture medium, and(ii) if the amount of manganese is below a target range, supplementingthe cell culture medium or the component of the cell culture medium withmanganese to achieve an amount of manganese within the target range,wherein the target range is sufficient to produce interferon beta-1apolypeptide with a predetermined glycosylation profile.

In another aspect, the invention is directed to a method for optimizinga cell culture medium for the production of an interferon beta-1apolypeptide comprising (i) determining the amount of manganese in a cellculture medium or a component used to produce a cell culture medium, and(ii) if the amount of manganese is above a target range, removingmanganese from the cell culture medium or the component of the cellculture medium to achieve an amount of manganese within the targetrange, wherein the target range is sufficient to produce interferonbeta-1a polypeptide with a predetermined glycosylation profile.

In one embodiment, the predetermined glycosylation profile of theinterferon beta-1a polypeptide comprises 91 to 100% sialylation. In afurther embodiment, the predetermined glycosylation profile of theinterferon beta-1a polypeptide comprises 91 to 97% sialylation. In afurther embodiment, the predetermined glycosylation profile of theinterferon beta-1a polypeptide comprises 95% sialylation.

In one embodiment, the predetermined glycosylation profile of theinterferon beta-1a polypeptide comprises 55 to 85% biantennaryglycoproteins. In a further embodiment, the predetermined glycosylationprofile of the interferon beta-1a polypeptide comprises 66 to 73%biantennary glycoproteins. In a further embodiment, the predeterminedglycosylation profile of the interferon beta-1a polypeptide comprises70% biantennary glycoproteins.

In one embodiment, the predetermined glycosylation profile of theinterferon beta-1a polypeptide comprises 12 to 46% triantennaryglycoproteins. In a further embodiment, the predetermined glycosylationprofile of the interferon beta-1a polypeptide comprises 26 to 35%triantennary glycoproteins. In a further embodiment, the predeterminedglycosylation profile of the interferon beta-1a polypeptide comprises30% triantennary glycoproteins.

In one embodiment, the predetermined glycosylation profile of theinterferon beta-1a polypeptide comprises a biantennary glycoprotein totriantennary glycoprotein ratio of about 1.8 to 4.6. In a furtherembodiment, the predetermined glycosylation profile of the interferonbeta-1a polypeptide comprises a biantennary glycoprotein to triantennaryglycoprotein ratio of about 2.0 to 2.5. In a further embodiment, thepredetermined glycosylation profile of the interferon beta-1apolypeptide comprises a biantennary glycoprotein to triantennaryglycoprotein ratio of about 2.3.

In one embodiment, the target manganese concentration range in the cellculture for interferon beta-1a polypeptide is 0.1 μM to 5 μM. In anotherembodiment, the target manganese concentration range in the cell culturefor interferon beta-1a polypeptide is 0.2 μM to 4.8 μM. In anotherembodiment, the target manganese concentration range in the cell culturefor interferon beta-1a polypeptide 0.3 μM to 4.8 μM.

In one embodiment, the target manganese concentration in the cellculture for anti-α4-integrin antibody or interferon beta-1a polypeptideis maintained through a feedback loop. In another embodiment, themanganese concentration is constantly monitored and maintained withinthe target manganese concentration range. In another embodiment, thetarget manganese concentration is achieved with a single dose ofmanganese.

In one aspect, the present invention is directed to a method forachieving a predetermined glycosylation profile of a rFVIIIFcpolypeptide comprising providing manganese to a cell culture at aconcentration that falls within a target manganese concentration range,wherein the cell culture comprises host cells producing the a rFVIIIFcpolypeptide. In another aspect, the invention is directed tosupplementing the cell culture with manganese if the manganeseconcentration in the cell culture is below the target manganeseconcentration range.

In another aspect, the invention is directed to a method for achieving apredetermined glycosylation profile of a rFVIIIFc polypeptide comprising(i) determining a manganese concentration in a component of a cellculture medium, (ii) if the manganese concentration is below a targetmanganese concentration range, supplementing the cell culture mediumwith the component to achieve a manganese concentration within thetarget manganese concentration range, and (iii) culturing a recombinanthost cell producing an a rFVIIIFc polypeptide in the cell culture mediumcomprising the cell culture medium component.

In another aspect, the invention is directed to a method for achieving apredetermined glycosylation profile of a rFVIIIFc polypeptide comprising(i) determining a manganese concentration in a component of a cellculture medium, (ii) if the manganese concentration is below a targetmanganese concentration range, adding manganese to the component of thecell culture medium to achieve a manganese concentration within thetarget manganese concentration range, (iii) producing a cell culturemedium using the component of cell culture medium with the targetmanganese concentration, and (iv) culturing a recombinant host cellproducing a rFVIIIFc polypeptide in the cell culture medium comprisingthe cell culture medium component with the target manganeseconcentration.

In another aspect, the invention is directed to a method for optimizinga cell culture medium for the production of a rFVIIIFc polypeptidecomprising (i) determining the amount of manganese in a cell culturemedium or a component used to produce a cell culture medium, and (ii) ifthe amount of manganese is below a target range, supplementing the cellculture medium or the component of the cell culture medium withmanganese to achieve an amount of manganese within the target range,wherein the target range is sufficient to produce a rFVIIIFc polypeptidewith a predetermined glycosylation profile.

In another aspect, the invention is directed to a method for optimizinga cell culture medium for the production of a rFVIIIFc polypeptidecomprising (i) determining the amount of manganese in a cell culturemedium or a component used to produce a cell culture medium, and (ii) ifthe amount of manganese is above a target range, removing manganese fromthe cell culture medium or the component of the cell culture medium toachieve an amount of manganese within the target range, wherein thetarget range is sufficient to produce a rFVIIIFc polypeptide with apredetermined glycosylation profile.

In one embodiment, the predetermined glycosylation profile of therFVIIIFc comprises 15 to 32% G0+Man6. In a further embodiment, thepredetermined glycosylation profile of the rFVIIIFc comprises 16 to 29%G0+Man6. In a further embodiment, the predetermined glycosylationprofile of the rFVIIIFc comprises 17 to 27% G0+Man6.

In one embodiment, the predetermined glycosylation profile of therFVIIIFc comprises 19 to 30% G1+Man7. In a further embodiment, thepredetermined glycosylation profile of the rFVIIIFc comprises 22 to 27%G1+Man7. In a further embodiment, the predetermined glycosylationprofile of the rFVIIIFc comprises 24 to 26% G1+Man7.

In one embodiment, the predetermined glycosylation profile of therFVIIIFc comprises 6 to 17% G2+Man9. In a further embodiment, thepredetermined glycosylation profile of the rFVIIIFc comprises 7 to 16%G2+Man9. In a further embodiment, the predetermined glycosylationprofile of the rFVIIIFc comprises 10 to 15% G2+Man9.

In one embodiment, the target manganese concentration range in the cellculture for a rFVIIIFc polypeptide is 30 nM to 1800 nM. In anotherembodiment, the target manganese concentration range in the cell culturefor a rFVIIIFc polypeptide is 50 nM to 300 nM. In another embodiment,the target manganese concentration range in the cell culture for arFVIIIFc polypeptide is 75 nM to 200 nM.

In one embodiment, the target manganese concentration in the cellculture for anti-α4-integrin antibody, or interferon beta-1apolypeptide, or a rFVIIIFc polypeptide is maintained through a feedbackloop. In another embodiment, the manganese concentration is constantlymonitored and maintained within the target manganese concentrationrange. In another embodiment, the target manganese concentration isachieved with a single dose of manganese.

In one embodiment, the anti-α4-integrin antibody, interferon beta-1apolypeptide, or rFVIIIFc polypeptide is produced by a eukaryotic hostcell. In a preferred embodiment, the eukaryotic host cell is a mammalianhost cell.

In one embodiment, the anti-α4-integrin antibody, interferon beta-1apolypeptide, or rFVIIIFc polypeptide is produced at a manufacturingscale. In another embodiment, the manganese concentration alters thelevels of the isoform variants of the anti-α4-integrin antibody,interferon beta-1a polypeptide, or rFVIIIFc polypeptide.

In one embodiment, the anti-α4-integrin antibody is natalizumab. In oneembodiment, the interferon beta-1a polypeptide is AVONEX® with SEQ IDNO:1

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A and FIG. 1B. FIG. 1A and FIG. 1B show the effect of manganeseconcentration on percent glycosylation of a recombinant interferonbeta-1a polypeptide. The X-axis shows manganese concentration of cellculture media. The Y-axis shows the percentage of glycans in recombinantinterferon beta-1a polypeptide. Specifically, the percent biantennary,percent TriLac, and the percent triantennary glycosylation of therecombinant interferon beta-1a polypeptide are shown in FIG. 1A andpercentage sialylation is shown in FIG. 1B.

FIG. 2. FIG. 2 shows the effect of manganese concentration on percentglycosylation of recombinant Factor VIII (rFVIIIFc). The X-axis showsmanganese concentration of cell culture media. The Y-axis shows thepercentage of glycosylation of a rFVIIIFc. Specifically, percentterminal mannose-6 with a G0 modification (G0+Man6), percent terminalmannose-7 with a G1 modification (G1+Man7), and percent terminalmannose-9 with a G2 modification (G2+Man9) is shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the recognition that cell culturemedia supplemented with manganese provides the ability to control andmanipulate the glycolsylation patterns of recombinant glycoproteinsproduced in eukaryotic cell cultures. Such glyclosylation patternsinclude, without limitation, the antennarity and level ofgalactosylation.

The present invention is also applicable to modifying the glycosylationof a recombinant glycoprotein of interest such that it falls within thequality attribute ranges for the desired product. For example, thepresent invention is applicable to modifying the glycosylation profileof a recombinant glycoprotein of interest to more closely resemble,match, or substantially match the glycosylation pattern of a referencesample of the same glycoprotein. Differences between variousmanufacturing processes can result in glycoproteins with identical aminoacid sequences having different glycosylation patterns depending on, forexample, conditions for growth, cell line used to express theglycoprotein, etc.

Provided herein are methods for achieving a predetermined glycosylationprofile of a recombinant glycoprotein of interest comprising adjustingthe concentration of manganese in a cell culture to achieve a targetconcentration range, wherein the cell culture comprises host cellsproducing the recombinant glycoprotein of interest. Also provided hereinare methods for optimizing a cell culture medium for the production of arecombinant glycoprotein of interest comprising (i) determining theamount of manganese in a cell culture medium or a component used toproduce a cell culture medium, and (ii) adjusting the concentration ofmanganese in the cell culture medium to achieve an amount of manganesewithin the target range, wherein the target range is sufficient toproduce the recombinant glycoprotein of interest with a predeterminedgalactosylation profile.

For example, provided herein are methods for achieving a predeterminedglycosylation profile of an anti-α4-integrin antibody, an interferonbeta-1a polypeptide, or a rFVIIIFc polypeptide comprising providingmanganese to a cell culture at a concentration that falls within atarget manganese concentration range, wherein the cell culture compriseshost cells producing the anti-α4-integrin antibody, an interferonbeta-1a polypeptide, or a rFVIIIFc polypeptide.

Also provide herein are methods for achieving a predeterminedglycosylation profile of an anti-α4-integrin antibody, an interferonbeta-1a polypeptide, or a rFVIIIFc polypeptide comprising (i)determining a manganese concentration in a component of a cell culturemedium, (ii) if the manganese concentration is below a target manganeseconcentration range, supplementing the cell culture medium with thecomponent to achieve a manganese concentration within the targetmanganese concentration range, and (iii) culturing a recombinant hostcell producing an anti-α4-integrin antibody, an interferon beta-1apolypeptide, or a rFVIIIFc polypeptide in the cell culture mediumcomprising the cell culture medium component.

Also provided herein are methods for achieving a predeterminedglycosylation profile of an anti-α4-integrin antibody, an interferonbeta-1a polypeptide, or a rFVIIIFc polypeptide comprising (i)determining a manganese concentration in a component of a cell culturemedium, (ii) if the manganese concentration is below a target manganeseconcentration range, adding manganese to the component of the cellculture medium to achieve a manganese concentration within the targetmanganese concentration range, (iii) producing a cell culture mediumusing the component of cell culture medium with the target manganeseconcentration, and (iv) culturing a recombinant host cell producing ananti-α4-integrin antibody, an interferon beta-1a polypeptide, or arFVIIIFc polypeptide in the cell culture medium comprising the cellculture medium component with the target manganese concentration.

Also provided herein are methods for optimizing a cell culture mediumfor the production of an anti-α4-integrin antibody, an interferonbeta-1a polypeptide, or a rFVIIIFc polypeptide comprising (i)determining the amount of manganese in a cell culture medium or acomponent used to produce a cell culture medium, and (ii) if the amountof manganese is above a target range, removing manganese from the cellculture medium or the component of the cell culture medium to achieve anamount of manganese within the target range, wherein the target range issufficient to produce anti-α4-integrin antibody, an interferon beta-1apolypeptide, or a rFVIIIFc polypeptide with a predeterminedglycosylation profile.

I. Definitions

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. The terms “a” (or “an”), as well as theterms “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever embodiments are described with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various embodimentsof the disclosure, which can be had by reference to the specification asa whole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The terms “polypeptide” or “protein” as used herein refers a sequentialchain of amino acids linked together via peptide bonds. The term is usedto refer to an amino acid chain of any length, but one of ordinary skillin the art will understand that the term is not limited to lengthychains and can refer to a minimal chain comprising two amino acidslinked together via a peptide bond. If a single polypeptide is thediscrete functioning unit and does require permanent physicalassociation with other polypeptides in order to form the discretefunctioning unit, the terms “polypeptide” and “protein” as used hereinare used interchangeably. If discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” as used herein refers to the multiple polypeptidesthat are physically coupled and function together as the discrete unit.

The term “glycoprotein” refers to a polypeptide or protein coupled to atleast one carbohydrate moiety, e.g., a polysaccharide or anoligosaccharide, that is attached to the protein via anoxygen-containing or a nitrogen-containing side chain of an amino acidresidue, e.g., a serine or threonine residue (“O-linked”) or anasparagine residue (“N-linked”). The term “glycan” refers to apolysaccharide or an oligosaccharide, e.g., a polymer comprised ofmonosaccharides. Glycans can be homo- or heteropolymers ofmonosaccharide residues, and can be linear or branched.

As used herein, the “glycosylation pattern” of a recombinantglycoprotein of interest refers to various physical characteristics ofthe glycoprotein's polysaccharides or oligosaccharides, such as, e.g.,the quantity and quality of various monosaccharides present, the degreeof branching, and/or the attachment (e.g., N-linked or O-linked). The“glycosylation pattern” of a glycoprotein can also refer to thefunctional characteristics imparted by the glycoprotein'soligosaccharides and polysaccharides. For example, the extent to whichthe glycoprotein can bind to FcγRIIIa and induce antibody-dependentcellular cytotoxicity (ADCC).

“Fucosylation” refers to the degree and distribution of fucose residueson polysaccharides and oligosaccharides, for example, N-glycans,0-glycans and glycolipids. Therapeutic glycoproteins, e.g., antibodiesor Fc fusion proteins, with non-fucosylated, or “afucosylated” N-glycansexhibit dramatically enhanced antibody-dependent cellular cytotoxicity(ADCC) due to the enhancement of FcγRIIIa binding capacity without anydetectable change in complement-dependent cytotoxicity (CDC) or antigenbinding capability. In certain situations, e.g., cancer treatment,non-fucosylated or “afucosylated” antibodies are desirable because theycan achieve therapeutic efficacy at low doses, while inducing highcellular cytotoxicity against tumor cells, and triggering high effectorfunction in NK cells via enhanced interaction with FcγRIIIa. In othersituations, e.g., treatment of inflammatory or autoimmune diseases,enhanced ADCC and FcγRIIIa binding is not desirable, and accordinglytherapeutic glycoproteins with higher levels of fucose residues in theirN-glycans can be preferable. As used herein, the term “% afucose” refersto the percentage of non-fucosylated N-glycans present on a recombinantglycoprotein of interest. A higher % afucose denotes a higher number ofnon-fucosylated N-glycans, and a lower % afucose denotes a higher numberof fucosylated N-glycans.

“Sialylation” refers to the type and distribution of sialic acidresidues on polysaccharides and oligosaccharides, for example,N-glycans, O-glycans and glycolipids. Sialic acids are most often foundat the terminal position of glycans. Sialylation can significantlyinfluence the safety and efficacy profiles of these proteins. Inparticular, the in vivo half-life of some biopharmaceuticals correlateswith the degree of oligosaccharide sialylation. Furthermore, thesialylation pattern can be a very useful measure of product consistencyduring manufacturing.

The two main types of sialyl residues found in biopharmaceuticalsproduced in mammalian expression systems are N-acetyl-neuraminic acid(NANA) and N-glycolylneuraminic acid (NGNA). These usually occur asterminal structures attached to galactose (Gal) residues at thenon-reducing terminii of both N- and O-linked glycans.

“Galactosylation” refers to the type and distribution of galactoseresidues on polysaccharides and oligosaccharides. Galactose refers to agroup of monosaccharides which include open chain and cyclic forms. Animportant disaccharide form of galactose isgalactose-alpha-1,3-galactose (α-gal).

“Antennarity” refers to the ratio of biantennary to triantennary ofpolysaccharides and oligosacchrides. The glycans in glycoproteins maydiffer by the number of branches making up the glycans, leading tobiantennary and triantennary structures.

The term “undesirable side effects” refers to certain aspects andresults of glycosylation which, under certain circumstances, are to beminimized or avoided. In certain aspects, a side effect to be reduced oravoided is a substantial increase in the level of α-gal. In anotheraspect a side effect to be reduced or avoided is a substantial reductionin sialic acid levels. In various aspects the methods described hereinachieve certain glycosylation patterns without substantially affectingculture density, cell viability level, or both. In certain aspects, a“side effect” which might be undesirable in one glycoprotein, e.g., adecrease in fucose levels (increases ADCC and FcγRIIIa binding) in anantibody used to treat an inflammatory disease, might be desirable inanother glycoprotein, e.g., in an antibody used to treat cancer.

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, monovalent or monospecific antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

“Recombinantly expressed glycoprotein” and “recombinant glycoprotein” asused herein refer to a glycoprotein expressed from a host cell that hasbeen genetically engineered to express that glycoprotein. Therecombinantly expressed glycoprotein can be identical or similar toglycoproteins that are normally expressed in the mammalian host cell.The recombinantly expressed glycoprotein can also be foreign to the hostcell, i.e. heterologous to peptides normally expressed in the mammalianhost cell. Alternatively, the recombinantly expressed glycoprotein canbe chimeric in that portions of the glycoprotein contain amino acidsequences that are identical or similar to glycoproteins normallyexpressed in the mammalian host cell, while other portions are foreignto the host cell. In certain embodiments, the recombinant glycoproteincomprises an antibody or fragments thereof. As used herein, the terms“recombinantly expressed glycoprotein” and “recombinant glycoprotein”also encompasses an antibody produced by a hybridoma.

The term “chimeric,” polypeptide or antibody as used herein, means apolypeptide or antibody that includes within it amino acid sequences (orportions thereof such as subsequences or peptides) from at least twodifferent sources, such as mouse and human. Chimeric polypeptides caninclude one or more linkers joining its portions. Chimeric polypeptidesor antibodies can include additional peptides such as signal sequencesand sequences such as 6His and FLAG that aid in protein purification ordetection.

The term “expression” or “expresses” are used herein to refer totranscription and translation occurring within a host cell. The level ofexpression of a product gene in a host cell can be determined on thebasis of either the amount of corresponding mRNA that is present in thecell or the amount of the protein encoded by the product gene that isproduced by the cell. For example, mRNA transcribed from a product geneis desirably quantitated by northern hybridization, Sambrook et al.,Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring HarborLaboratory Press, 1989). Protein encoded by a product gene can bequantitated either by assaying for the biological activity of theprotein or by employing assays that are independent of such activity,such as western blotting or radioimmunoassay using antibodies that arecapable of reacting with the protein, Sambrook et al., MolecularCloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring HarborLaboratory Press, 1989).

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 can be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of a moleculeof interest may be assessed in vivo, e.g., in an animal model such asthat disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “basal media formulation” or “basal media” as used hereinrefers to any cell culture media used to culture cells that has not beenmodified either by supplementation, or by selective removal of a certaincomponent.

The term “component” refers to an ingredient or a part of an additive orcell culture medium.

As used herein, the terms “additive” or “supplement” refer to anysupplementation made to a basal medium to achieve the goals described inthis disclosure. An “additive” or “supplement” can include a singlesubstance, e.g., manganese chloride, or can include multiple substances,e.g., various manganese salts. The terms “additive” or “supplement”refer to the all of the components added, even though they need not beadded at the same time, and they need not be added in the same way. Forexample, one or more components of an “additive” or “supplement” can beadded as a single bolus or two or more boli from a stock solution, whileother components of the same “additive” or “supplement” can be added aspart of a feed medium. In addition, any one or more components of an“additive” or “supplement” can be present in the basal medium from thebeginning of the cell culture.

The terms “culture”, “cell culture” and “eukaryotic cell culture” asused herein refer to a eukaryotic cell population, eithersurface-attached or in suspension that is maintained or grown in amedium (see definition of “medium” below) under conditions suitable tosurvival and/or growth of the cell population. As will be clear to thoseof ordinary skill in the art, these terms as used herein can refer tothe combination comprising the mammalian cell population and the mediumin which the population is suspended.

The terms “media”, “medium”, “cell culture medium”, “culture medium”,“tissue culture medium”, “tissue culture media”, and “growth medium” asused herein refer to a solution containing nutrients, which nourishgrowing cultured eukaryotic cells. Typically, these solutions provideessential and non-essential amino acids, vitamins, energy sources,lipids, and trace elements required by the cell for minimal growthand/or survival. The solution can also contain components that enhancegrowth and/or survival above the minimal rate, including hormones andgrowth factors. The solution is formulated to a pH and saltconcentration optimal for cell survival and proliferation. The mediumcan also be a “defined medium” or “chemically defined medium”—aserum-free medium that contains no proteins, hydrolysates or componentsof unknown composition. Defined media are free of animal-derivedcomponents and all components have a known chemical structure. One ofskill in the art understands a defined medium can comprise recombinantglycoproteins or proteins, for example, but not limited to, hormones,cytokines, interleukins and other signaling molecules.

The cell culture medium is generally “serum free” when the medium isessentially free of serum, or fractions thereof, from any mammaliansource (e.g. fetal bovine serum (FBS)). By “essentially free” is meantthat the cell culture medium comprises between about 0-5% serum,preferably between about 0-1% serum, and most preferably between about0-0.1% serum. Advantageously, serum-free “defined” medium can be used,wherein the identity and concentration of each of the components in themedium is known (i.e., an undefined component such as bovine pituitaryextract (BPE) is not present in the culture medium).

The term “cell viability” as used herein refers to the ability of cellsin culture to survive under a given set of culture conditions orexperimental variations. The term as used herein also refers to thatportion of cells which are alive at a particular time in relation to thetotal number of cells, living and dead, in the culture at that time.

The term “cell density” as used herein refers to that number of cellspresent in a given volume of medium.

The term “batch culture” as used herein refers to a method of culturingcells in which all the components that will ultimately be used inculturing the cells, including the medium (see definition of “medium”below) as well as the cells themselves, are provided at the beginning ofthe culturing process. A batch culture is typically stopped at somepoint and the cells and/or components in the medium are harvested andoptionally purified.

The term “fed-batch culture” as used herein refers to a method ofculturing cells in which additional components are provided to theculture at some time subsequent to the beginning of the culture process.A fed-batch culture can be started using a basal medium. The culturemedium with which additional components are provided to the culture atsome time subsequent to the beginning of the culture process is a feedmedium. A fed-batch culture is typically stopped at some point and thecells and/or components in the medium are harvested and optionallypurified.

The term “perfusion culture” as used herein refers to a method ofculturing cells in which additional components are provided continuouslyor semi-continuously to the culture subsequent to the beginning of theculture process. The provided components typically comprise nutritionalsupplements for the cells which have been depleted during the culturingprocess. A portion of the cells and/or components in the medium aretypically harvested on a continuous or semi-continuous basis and areoptionally purified.

The term “bioreactor” as used herein refers to any vessel used for thegrowth of a mammalian cell culture. The bioreactor can be of any size solong as it is useful for the culturing of mammalian cells. Typically,the bioreactor will be at least 1 liter and can be 10, 50, 100, 250,500, 1000, 2000, 2500, 3000, 5000, 8000, 10,000, 12,0000, 15,000,20,000, 30,000 liters or more, or any volume in between. For example, abioreactor will be 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000liters, 10 to 20,000 liters, 10 to 30,000 liters, 50 to 5,000 liters, 50to 10,000 liters, 50 to 15,000 liters, 50 to 20,000 liters, 50 to 30,000liters, 1,000 to 5,000 liters, or 1,000 to 3,000 liters. A bioreactorcan be a stirred-tank bioreactor or a shake flask. The internalconditions of the bioreactor, for example, but not limited to pH andtemperature, are typically controlled during the culturing period. Thebioreactor can be composed of any material that is suitable for holdingmammalian cell cultures suspended in media under the culture conditionsof the present invention, including glass, plastic or metal. The term“production bioreactor” as used herein refers to the final bioreactorused in the production of the glycoprotein or protein of interest. Thevolume of the large-scale cell culture production bioreactor istypically at least 500 liters and can be 1000, 2000, 2500, 5000, 8000,10,000, 12,0000, 15,000 liters or more, or any volume in between. Forexample, the large scale cell culture reactor will be between about 500liters and about 20,000 liters, about 500 liters and about 10,000liters, about 500 liters and about 5,000 liters, about 1,000 liters andabout 30,000 liters, about 2,000 liters and about 30,000 liters, about3,000 liters and about 30,000 liters, about 5,000 liters and about30,000 liters, or about 10,000 liters and about 30,000 liters, or alarge scale cell culture reactor will be at least about 500 liters, atleast about 1,000 liters, at least about 2,000 liters, at least about3,000 liters, at least about 5,000 liters, at least about 10,000 liters,at least about 15,000 liters, or at least about 20,000 liters. One ofordinary skill in the art will be aware of and will be able to choosesuitable bioreactors for use in practicing the present invention.

The term “stirred-tank bioreactor” as used herein refers to any vesselused for the growth of a mammalian cell culture that has an impeller.

The term “shake flask” as used herein refers to any vessel used for thegrowth of a mammalian cell culture that does not have an impeller.

The term “hybridoma” as used herein refers to a cell created by fusionof an immortalized cell derived from an immunologic source and anantibody-producing cell. The resulting hybridoma is an immortalized cellthat produces antibodies. The individual cells used to create thehybridoma can be from any mammalian source, including, but not limitedto, rat, pig, rabbit, sheep, pig, goat, and human. The term alsoencompasses trioma cell lines, which result when progeny of heterohybridmyeloma fusions, which are the product of a fusion between human cellsand a murine myeloma cell line, are subsequently fused with a plasmacell. Furthermore, the term is meant to include any immortalized hybridcell line that produces antibodies such as, for example, quadromas (See,e.g., Milstein et al., Nature, 537:3053 (1983)).

The term “osmolality” is a measure of the osmotic pressure of dissolvedsolute particles in an aqueous solution. The solute particles includeboth ions and non-ionized molecules. Osmolality is expressed as theconcentration of osmotically active particles (i.e., osmoles) dissolvedin 1 kg of water (1 mOsm/kg H₂O at 38° C. is equivalent to an osmoticpressure of 19 mm Hg). “Osmolarity” refers to the number of soluteparticles dissolved in 1 liter of solution. Solutes which can be addedto the culture medium so as to increase the osmolality thereof includeproteins, peptides, amino acids, non-metabolized polymers, vitamins,ions, salts, sugars, metabolites, organic acids, lipids, etc. In thepreferred embodiment, the concentration of amino acids and NaCl in theculture medium is increased in order to achieve the desired osmolalityranges set forth herein. When used herein, the abbreviation “mOsm” means“milliosmoles/kg H₂O”.

The term “titer” as used herein refers to the total amount ofrecombinantly expressed glycoprotein or protein produced by a cellculture divided by a given amount of medium volume. Titer is typicallyexpressed in units of milligrams of glycoprotein or protein permilliliter of medium or in units of grams of glycoprotein or protein perliter of medium.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., cellular viability). Thedifference between said two values is, for example, less than about 50%,less than about 40%, less than about 30%, less than about 20%, and/orless than about 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein with regard to amounts or numerical values (and not asreference to the chemical process of reduction), denotes a sufficientlyhigh degree of difference between two numeric values (generally oneassociated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., cellular viability). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

II. Supplementation of Cell Culture Medium to Control GlycosylationPatterns

Provided herein are methods to culture eukaryotic cells engineered toexpress a recombinant glycoprotein of interest. Specifically thisdisclosure provides methods for controlling the glycosylation patternsof a recombinant glycoprotein of interest by supplementing a tissueculture medium in which the cells are growing and/or producing therecombinant glycoprotein of interest with an additive, or culturingeukaryotic cells engineered to express a glycoprotein of interest in atissue culture medium, which has been supplemented with such anadditive. In certain embodiments, glycoproteins produced by the methodsprovided are recovered. The methods are based on the recognition thatgrowth of cells expressing a recombinant glycoprotein of interest incell culture medium supplemented with manganese can result inalterations to eukaryotic cell glycosylation patterns, such as the levelof galactosylation. In certain embodiments, the manganese added ismanganese chloride. In certain embodiments, the alteration of theglycosylation pattern of the recombinant glycoprotein of interestcomprises a reduced level of galactosylation.

In one embodiment, the recombinant glycoprotein of interest comprises apredetermined galactosylation profile. In one embodiment, therecombinant glycoprotein of interest comprises a predeterminedfucosylation profile. In one embodiment, the recombinant glycoprotein ofinterest comprises a predetermined mannosylation profile. In oneembodiment, the recombinant glycoprotein of interest comprises apredetermined sialylation profile. In one embodiment, the recombinantglycoprotein of interest comprises a predetermined % biantennaryglycoproteins profile. In one embodiment, the recombinant glycoproteinof interest comprises a predetermined % biantennary glycoproteinsprofile. In one embodiment, the recombinant glycoprotein of interestcomprises a predetermined % triantennary glycoproteins profile. In oneembodiment, the recombinant glycoprotein of interest comprises apredetermined biantennary glycoprotein to triantennary glycoproteinratio.

In one embodiment, the recombinant glycoprotein of interest comprising apredetermined glycosylation profile is an anti-α4-integrin antibody. Inanother embodiment, the recombinant glycoprotein of interest comprisinga predetermined glycosylation profile is an interferon beta-1apolypeptide. In another embodiment, the recombinant glycoprotein ofinterest comprising a predetermined glycosylation profile is a rFVIIIFcpolypeptide.

In another embodiment, the predetermined galactosylation profile of theanti-α4-integrin antibody comprises 10 to 35% galactosylation, 13 to 32%galactosylation, 15 to 32% galactosylation, 16 to 30% galactosylation,18 to 28% galactosylation, 18 to 31% galactosylation, 20 to 27%galactosylation, or 20 to 24% galactosylation. In another embodiment,the predetermined galactosylation profile of the anti-α4-integrinantibody comprises 13 to 32% or 18 to 31% galactosylation. In anotherembodiment, the predetermined galactosylation profile of theanti-α4-integrin antibody comprises about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,or 35% galactosylation. In another embodiment, the predeterminedgalactosylation profile of the anti-α4-integrin antibody comprises X %galactosylation.

In another embodiment, the predetermined sialylation profile of theanti-α4-integrin antibody comprises 0.1 to 5% sialylation, 0.5 to 4.5%sialylation, 0.7 to 3.6% sialylation, 1.0 to 3.0% sialylation, 1.0 to2.2% sialylation, X or 1.5 to 2.0% sialylation. In another embodiment,the predetermined sialylation profile of the anti-α4-integrin antibodycomprises 0.7 to 3.6% or 1.0 to 2.2% sialylation. In another embodiment,the predetermined sialylation profile of the anti-α4-integrin antibodycomprises about 0.1, 0.2, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8,2.0, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, 3.5, 3.6, 3.8, 4.0, 4.2,4.4, 4.5, 4.8, or 5.0% sialylation. In another embodiment, thepredetermined sialylation profile of the anti-α4-integrin antibodycomprises 1.6% sialylation.

In another embodiment, the predetermined sialylation profile ofinterferon beta-1a polypeptide comprises 75 to 100% sialylation, 80 to100% sialylation, 95 to 100% sialylation, 85 to 100% sialylation, 91 to100% sialylation, 75 to 97% sialylation, 80 to 97% sialylation, 85 to97%, 91 to 97% sialylation, or 95 to 97% sialylation. In anotherembodiment, the predetermined sialylation profile of interferon beta-1apolypeptide comprises 91 to 100% or 91 to 97% sialylation. In anotherembodiment, the predetermined sialylation profile of interferon beta-1apolypeptide comprises about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% sialylation. In another embodiment, the predeterminedsialylation profile of interferon beta-1a polypeptide comprises 95%sialylation.

In another embodiment, the predetermined % biantennary glycoproteinsprofile of the interferon beta-1a polypeptide comprises 50 to 100%biantennary glycoproteins, 50 to 90% biantennary glycoproteins, 50 to85% biantennary glycoproteins, 50 to 80% biantennary glycoproteins, 50to 75% biantennary glycoproteins, 50 to 60% biantennary glycoproteins,55 to 85% biantennary glycoproteins, 66 to 73% biantennaryglycoproteins, 55 to 80% biantennary glycoproteins, or 66 to 70%biantennary glycoproteins. In another embodiment, the predetermined %biantennary glycoproteins profile of the beta-1a polypeptide comprises55 to 85% or 66 to 73% biantennary glycoproteins. In another embodiment,the predetermined % biantennary glycoproteins profile of the beta-1apolypeptide comprises about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, or 90% biantennary glycoproteins. In anotherembodiment, the predetermined % biantennary glycoproteins profile of theanti-α4-integrin antibody comprises 70% biantennary glycoproteins.

In another embodiment, the predetermined % triantennary glycoproteinsprofile of interferon beta-1a polypeptide comprises 10 to 50%triantennary glycoproteins, 10 to 45% triantennary glycoproteins, 12 to46% triantennary glycoproteins, 10 to 35% triantennary glycoproteins, 20to 35% triantennary glycoproteins, 26 to 35% triantennary glycoproteins,10 to 35% triantennary glycoproteins, or 26 to 50% triantennaryglycoproteins. In another embodiment, the predetermined % triantennaryglycoproteins profile of interferon beta-1a polypeptide comprises 12 to46% or 26 to 35% triantennary glycoproteins. In another embodiment, thepredetermined % triantennary glycoproteins profile of interferon beta-1apolypeptide comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35%triantennary glycoproteins. In another embodiment, the predetermined %triantennary glycoproteins profile of interferon beta-1a polypeptidecomprises X % triantennary glycoproteins.

In another embodiment, the predetermined glycoproteins profile ofinterferon beta-1a polypeptide comprises biantennary glycoprotein totriantennary glycoprotein ratio of about 1.5 to 5, 1.8 to 5, 1.8 to 4.6,1.5 to 4, 1.5 to 3.5, 1.5 to 3, 1.5 to 2.5, 2.0 to 2.5, 2.0 to 3, 2.5 to3.5, 2.5 to 4, 2.5 to 4.5, 2.5 to 5.0, 3.0 to 4.0, 3.0 to 5.0, 3.0 to5.0, 3.5 to 4.0, or 3.5 to 5.0. In another embodiment, the predeterminedglycoproteins profile of interferon beta-1a polypeptide comprisesbiantennary glycoprotein to triantennary glycoprotein ratio of about 1.8to 4.6 or about 2.0 to 2.5. In another embodiment, the predeterminedglycoproteins profile of interferon beta-1a polypeptide comprisesbiantennary glycoprotein to triantennary glycoprotein ratio of about1.5, 1.8, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5,4.0, 4.5, or 5.0. In another embodiment, the predetermined glycoproteinsprofile of interferon beta-1a polypeptide comprises biantennaryglycoprotein to triantennary glycoprotein ratio of about 2.3.

In another embodiment, the predetermined glycosylation profile of therFVIIIFc comprises 15 to 32% G0+Man6, 10 to 40% G0+Man6, 16 to 29%G0+Man6, 20 to 30% G0+Man6, 25 to 30% G0+Man6, 17 to 27% G0+Man6, 25 to40% G0+Man6, or 15 to 20% G0+Man6. In another embodiment, thepredetermined G0+Man6 profile of the rFVIIIFc comprises 15 to 32%, 16 to29, or 17 to 27% G0+Man6. In another embodiment, the predeterminedglycosylation profile of the rFVIIIFc comprises about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40% G0+Man6.

In another embodiment, the predetermined glycosylation profile of therFVIIIFc comprises 10 to 40% G0+Man6, 19 to 30% G1+Man7, 15 to 40%G1+Man7, 20 to 40% G1+Man7, 25 to 40% G1+Man7, 30 to 40% G1+Man7, 10 to30% G1+Man7, 20 to 30% G1+Man7, 22 to 27% G1+Man7, or 24 to 26% G1+Man7.In another embodiment, the predetermined G1+Man7 profile of the rFVIIIFccomprises 19 to 30, 22 to 27% or 24-26% G1+Man7. In another embodiment,the predetermined glycosylation profile of the rFVIIIFc comprises about10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40% G1+Man7.

In another embodiment, the predetermined glycosylation profile of therFVIIIFc comprises 1 to 20% G2+Man9, 6 to 17% G2+Man9, 5 to 20% G2+Man9,10 to 20% G2+Man9, 7 to 16% G2+Man9, 15 to 20% G2+Man9, 10 to 15%G2+Man9, or 5 to 15% G2+Man9. In another embodiment, the predeterminedG2+Man9 profile of the rFVIIIFc comprises 6 to 17, 7 to 16% or 10-15%G2+Man9. In another embodiment, the predetermined glycosylation profileof the rFVIIIFc comprises about 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20% G2+Man9.

In one embodiment, the manganese concentration in the cell culturealters the levels of the isoform variants of the interferon beta-1apolypeptide. In one embodiment, the manganese concentration in the cellculture alters the level of an isoform of the interferon beta-1apolypeptide.

The present invention is applicable to altering, manipulating, orcontrolling the glycosylation pattern of a recombinant glycoprotein ofinterest to match, substantially match, approach, or more closelyresemble the glycosylation pattern of the same glycoprotein, butproduced in a different cell culture system. Recombinant glycoproteinsof interest can be produced according to the invention using variousdifferent cell culture systems, e.g., a batch culture, fed-batch culturea perfusion culture, a shake flask, and/or a bioreactor. In oneembodiment, cells expressing a recombinant glycoprotein of interest arecultured in basal medium to which the additive is introduced as a bolus,or two or more boli, from a stock solution. In another embodiment, theadditive is introduced as a component of a feed medium. In certainembodiments the cell culture comprises a growth phase and a proteinproduction phase, and the additive is introduced into the culture mediumbefore, or at the same time as, or at some point after the initiation ofthe protein production phase.

In one embodiment, a medium described herein is a serum-free medium,animal protein-free medium or a chemically-defined medium. In a specificembodiment, a medium described herein is a chemically-defined medium.

In certain embodiments, the method comprises adding MnCl₂. MnCl₂ can beadded to the culture medium in one bolus or two or more boli from astock solution to, or be added as a component of a feed medium achieve aMnCl₂ concentration in the culture medium of between about 0.025 μM toabout 10 μM MnCl₂ for anti-α4-integrin antibody. In certain embodimentsthe additive comprises MnCl₂, which can be added to the culture mediumin one bolus or two or more boli from a stock solution, or be added as acomponent of a feed medium to achieve a MnCl₂ concentration in theculture medium between about 0.025 μM and about 10 μM, about 0.5 μM andabout 10 μM, about 0.5 μM and about 7.5 μM, about 0.5 μM and about 5 μM,about 0.5 μM and about 2.5 μM, about 0.5 μM and about 1 μM, about 1 μMand about 10 μM, about 1 μM and about 10 μM, about 0.5 μM and about 7.5μM, about 1 μM and about 5 μM, about 0.1 μM and about 2.5 μM, about 0.2μM and about 2.5 μM, about 0.5 μM and about 2.5 μM, about 1 μM and about2.5 μM, about 0.2 μM and about 2.0 μM, about 0.2 μM and about 1.5 μM,about 2.5 μM and about 10 μM, about 2.5 μM and about 7.5 μM, about 2.5μM and about 5 μM, about 5 μM and about 10 μM, about 5 μM and about 7.5μM or about 7.5 μM and about 10 μM for anti-α4-integrin antibody.

In another embodiment, the target manganese concentration in the culturemedium is between 0.002 μM and 0.4 μM at Day 0 for anti-α4-integrinantibody. In another embodiment, the target manganese concentration inthe culture mediums is between 0.04 μM and 1 at Day 13 foranti-α4-integrin antibody. In another embodiment, the manganeseconcentration is constantly monitored and maintained within the targetmanganese concentration range. In another embodiment, the targetconcentration is maintained through a feedback loop.

In certain embodiments, the method comprises adding MnCl₂. MnCl₂ can beadded to the culture medium in one bolus or two or more boli from astock solution to, or be added as a component of a feed medium achieve aMnCl₂ concentration in the culture medium of between about 0.1 μM toabout 5 μM MnCl₂ for interferon beta-1a polypeptide. In certainembodiments the additive comprises MnCl₂, which can be added to theculture medium in one bolus or two or more boli from a stock solution,or be added as a component of a feed medium to achieve a MnCl₂concentration in the culture medium between about 0.1 μM and about 5 μM,about 0.1 μM and about 4 μM, about 0.1 μM and about 3.5 μM, about 0.1 μMand about 3.0 μM, about 0.1 μM and about 2.5 μM, about 0.1 μM and about2.0 μM, about 0.1 μM and about 1.5 μM, about 0.1 μM and about 1 μM,about 0.1 μM and about 0.5 μM, 0.2 μM and about 5 μM, about 0.2 μM andabout 4 μM, about 0.2 μM and about 3.5 μM, about 0.2 μM and about 3.0μM, about 0.2 μM and about 2.5 μM, about 0.2 μM and about 2.0 μM, about0.2 μM and about 1.5 μM, about 0.2 μM and about 1 μM, about 0.2 μM andabout 0.5 μM, 0.3 μM and about 5 μM, about 0.3 μM and about 4 μM, about0.3 μM and about 3.5 μM, about 0.3 μM and about 3.0 μM, about 0.3 μM andabout 2.5 μM, about 0.3 μM and about 2.0 μM, about 0.3 μM and about 1.5μM, about 0.3 μM and about 1 μM, about 0.3 μM and about 0.5 μM about 0.5μM and about 5 μM, about 0.5 μM and about 4 μM, about 0.5 μM and about 3μM, about 0.5 μM and about 2.5 μM, about 0.5 μM and about 2.0 μM, about0.5 μM and about 1.5 μM, about 0.5 μM and about 1 μM, about 1 μM andabout 5 μM, about 1 μM and about 4 μM about, about 1 μM and about 3 μM,1 μM and about 2.5 μM, about 2.5 μM and about 5 μM, about 0.2 μM toabout 4.8 μM, or 0.3 μM to about 4.8 μM for interferon beta-1apolypeptide.

In certain embodiments, the method comprises adding MnCl₂. MnCl₂ can beadded to the culture medium in one bolus or two or more boli from astock solution to, or be added as a component of a feed medium achieve aMnCl₂ concentration in the culture medium of between about 30 nM to 1800nM MnCl₂ for rFVIIIFc. In certain embodiments the additive comprisesMnCl₂, which can be added to the culture medium in one bolus or two ormore boli from a stock solution, or be added as a component of a feedmedium to achieve a MnCl₂ concentration in the culture medium betweenabout 25 nM to 1800 nM, 25 nM to 1500 nM, 30 nM to 1800 nM, 30 nM to1500 nM, about 30 nM to 1000 nM, about 30 nM to 800 nM, about 30 nM to500 nM, about 30 nM to 300 nM, 30 nM to 200 nM, about 30 nM to 100 nM,50 nM to 1500 nM, about 50 nM to 1000 nM, about 50 nM to 800 nM, about50 nM to 500 nM, about 50 nM to 300 nM, 50 nM to 200 nM, about 50 nM to100 nM, 75 nM to 1500 nM, about 75 nM to 1000 nM, about 75 nM to 800 nM,about 75 nM to 500 nM, about 75 nM to 300 nM, 75 nM to 200 nM, about or75 nM to 100 nM for rFVIIIFc.

In another embodiment, the manganese concentration is constantlymonitored and maintained within the target manganese concentrationrange. In another embodiment, the target concentration is maintainedthrough a feedback loop.

III. Cell Culture Compositions

The present invention further provides a cell culture compositioncomprising a medium described herein and cells, produced by the methodsprovided herein.

In one embodiment, a cell culture composition produced by the providedmethods can be a batch culture, fed-batch culture or a perfusionculture. In a specific embodiment, a cell culture composition of theinvention is a fed batch culture.

In one embodiment, a cell culture composition produced by the providedmethods comprises eukaryotic cells. In another embodiment, a cellculture composition produced by the provided methods comprises mammaliancells selected from the group consisting of CHO cells, HEK cells, NSOcells, PER.C6 cells, 293 cells, HeLa cells, and MDCK cells. In aspecific embodiment, a cell culture composition described hereincomprises CHO cells. In another specific embodiment, a cell culturecomposition described herein comprises HEK cells. In another specificembodiment, a cell culture composition described herein compriseshybridoma cells.

A cell culture composition produced by the provided methods can comprisecells that have been adapted to grow in serum free medium, animalprotein free medium or chemically defined medium. Or it can comprisecells that have been genetically modified to increase their life-span inculture. In one embodiment, the cells have been modified to express ananti-α4-integrin antibody. In a further embodiment, the cells have beenmodified to express natalizumab.

The present invention provides a method of culturing cells, comprisingcontacting the cells with a medium disclosed herein, supplementing themedium as described above, or culturing cells in a medium supplementedas described above.

Cell cultures can be cultured in a batch culture, fed batch culture or aperfusion culture. In one embodiment, a cell culture according to amethod of the present invention is a batch culture. In anotherembodiment, a cell culture according to a method of the presentinvention is a fed batch culture. In a further embodiment, a cellculture according to a method of the present invention is a perfusionculture. In certain embodiments the cell culture is maintained in ashake flask, in certain embodiments the cell culture is maintained in abioreactor.

In one embodiment, a cell culture according to a method of the presentinvention is a serum-free culture. In another embodiment, a cell cultureaccording to a method of the present invention is a chemically definedculture. In a further embodiment, a cell culture according to a methodof the present invention is an animal protein free culture.

In one embodiment, a cell culture produced by the provided methods iscontacted with a medium described herein during the growth phase of theculture. In another embodiment, a cell culture is contacted with amedium described herein during the production phase of the culture.

In one embodiment, a cell culture produced by the provided methods iscontacted with a feed medium described herein during the productionphase of the culture. In one embodiment, the culture is supplementedwith the feed medium between about 1 and about 25 times during thesecond time period. In another embodiment, a culture is supplementedwith the feed medium between about 1 and about 20 times, between about 1and about 15 times, or between about 1 and about 10 times during thefirst time period. In a further embodiment, a culture is supplementedwith the feed medium at least once, at least twice, at least threetimes, at least four times, at least five times, at least 6 times, atleast 7 times, at least 8 times, at least 9 times, at least 10 times, atleast 11 times, at least 12 times, at least 13 times, at least 14 times,at least 15 times, at least 20 times, at least 25 times. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

A culture produced by the provided methods can be contacted with a feedmedium described herein at regular intervals. In one embodiment, theregular interval is about once a day, about once every two days, aboutonce every three days, about once every 4 days, or about once every 5days. In a specific embodiment, the culture is a fed batch culture. Inanother specific embodiment, the culture is a perfusion culture.

A culture produced by the provided methods can be contacted with a feedmedium described herein on an as needed basis based on the metabolicstatus of the culture. In one embodiment, a metabolic marker of a fedbatch culture is measured prior to supplementing the culture with a feedmedium described herein. In one embodiment, the metabolic marker isselected from the group consisting of: glucose concentration, lactateconcentration, ammonium concentration, alanine concentration, glutamineconcentration, glutamate concentration, cell specific lactate productionrate to the cell specific glucose uptake rate ratio (LPR/GUR ratio), andRhodamine 123 specific cell fluorescence. In one embodiment, an LPR/GURvalue of >0.1 indicates the need to supplement the culture with a feedmedium described herein. In a further specific embodiment, a lactateconcentration of >3 g/L indicates the need to supplement the culturewith a feed medium described herein. In another embodiment, a cultureaccording to the present invention is supplemented with a feed mediumdescribed herein when the LPR/GUR value of the culture is >0.1 or whenthe lactate concentration of the culture is >3 g/L. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

In one embodiment, a medium described herein is a feed medium for a fedbatch cell culture. A skilled artisan understands that a fed batch cellculture can be contacted with a feed medium more than once. In oneembodiment, a fed batch cell culture is contacted with a mediumdescribed herein only once. In another embodiment, a fed batch cellculture is contacted with a medium described herein more than once, forexample, at least twice, at least three times, at least four times, atleast five times, at least six times, at least seven times, or at leastten times.

In accordance with the present invention, the total volume of feedmedium added to a cell culture should optimally be kept to a minimalamount. For example, the total volume of the feed medium added to thecell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45 or 50% of the volume of the cell culture prior to adding the feedmedium.

Cell cultures produced by the provided methods can be grown to achieve aparticular cell density, depending on the needs of the practitioner andthe requirement of the cells themselves, prior to being contacted with amedium described herein. In one embodiment, the cell culture iscontacted with a medium described herein at a viable cell density of 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 99 percent of maximal viable cell density. In a specificembodiment, the medium is a feed medium.

Cell cultures produced by the provided methods can be allowed to growfor a defined period of time before they are contacted with a mediumdescribed herein. In one embodiment, the cell culture is contacted witha medium described herein at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 of the cell culture. In another embodiment, the cell culture iscontacted with a medium described herein at week 1, 2, 3, 4, 5, 6, 7, or8 of the cell culture. In a specific embodiment, the medium is a feedmedium.

Cell cultures produced by the provided methods can be cultured in theproduction phase for a defined period of time. In one embodiment, thecell culture is contacted with a feed medium described herein at day 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the production phase.

A culture produced by the provided methods can be maintained inproduction phase for between about 1 day and about 30 days. In oneembodiment, a culture is maintained in production phase for betweenabout 1 day and about 30 days, between about 1 day and about 25 days,between about 1 day and about 20 days, about 1 day and about 15 days,about 1 day and about 14 days, about 1 day and about 13 days, about 1day and about 12 days, about 1 day and about 11 days, about 1 day andabout 10 days, about 1 day and about 9 days, about 1 day and about 8days, about 1 day and about 7 days, about 1 day and about 6 days, about1 day and about 5 days, about 1 day and about 4 days, about 1 day andabout 3 days, about 2 days and about 25 days, about 3 days and about 25days, about 4 days and about 25 days, about 5 days and about 25 days,about 6 days and about 25 days, about 7 days and about 25 days, about 8days and about 25 days, about 9 days and about 25 days, about 10 daysand about 25 days, about 15 days and about 25 days, about 20 days andabout 25 days, about 2 days and about 30 days, about 3 days and about 30days, about 4 days and about 30 days, about 5 days and about 30 days,about 6 days and about 30 days, about 7 days and about 30 days, about 8days and about 30 days, about 9 days and about 30 days, about 10 daysand about 30 days, about 15 days and about 30 days, about 20 days andabout 30 days, or about 25 days and about 30 days. In anotherembodiment, a culture is maintained in production phase for at leastabout 1 day, at least about 2 days, at least about 3 days, at leastabout 4 days, at least about 5 days, at least about 6 days, at leastabout 7 days, at least about 8 days, at least about 9 days, at leastabout 10 days, at least about 11 days, at least about 12 days, at leastabout 15 days, at least about 20 days, at least about 25 days, or atleast about 30 days. In a further embodiment, a culture is maintained inproduction phase for about 1 day, about 2 days, about 3 days, about 4days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 15 days, about20 days, about 25 days, or about 30 days.

The present invention further provides a method of producing arecombinant glycoprotein interest, comprising culturing cells engineeredto express the recombinant glycoprotein of interest in a culturecomprising a medium described herein; and recovering or isolating therecombinant glycoprotein of interest from the culture. In certainembodiments, the recombinant glycoprotein of interest is an antibody ora fragment thereof. In a specific embodiment, the recombinantglycoprotein of interest is an anti-α4-integrin antibody. In anotherembodiment, the recombinant glycoprotein of interest is natalizumab.

In a specific embodiment, a method of producing a recombinantglycoprotein of interest according to the present invention produces amaximum glycoprotein titer of at least about 0.05 g/L, at least about0.1 g/L, at least about 0.25 g/L, at least about 0.5 g/L, at least about0.75 g/L, at least about 1.0 g/L, at least about 1.5 g/L, at least about2 g/liter, at least about 2.5 g/liter, at least about 3 g/liter, atleast about 3.5 g/liter, at least about 4 g/liter, at least about 4.5g/liter, at least about 5 g/liter, at least about 6 g/liter, at leastabout 7 g/liter, at least about 8 g/liter, at least about 9 g/liter, orat least about 10 g/liter. In another embodiment, the method accordingto the present invention produces a maximum glycoprotein titer ofbetween about 1 g/liter and about 10 g/liter, about 1.5 g/liter andabout 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter,about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about4.5 g/liter, or about 1 g/liter and about 4 g/liter. In a specificembodiment, the glycoprotein is an antibody. In another embodiment, theglycoprotein is a blood clotting factor.

The invention further provides a conditioned cell culture mediumproduced by a method described herein.

In one embodiment, a conditioned cell culture medium produced accordingto the provided methods comprises a recombinant glycoprotein ofinterest. In a specific embodiment, a conditioned cell culture mediumaccording to the invention comprises a recombinant glycoprotein ofinterest at a titer of at least about 2 g/liter, at least about 2.5g/liter, at least about 3 g/liter, at least about 3.5 g/liter, at leastabout 4 g/liter, at least about 4.5 g/liter, at least about 5 g/liter,at least about 6 g/liter, at least about 7 g/liter, at least about 8g/liter, at least about 9 g/liter, or at least about 10 g/liter, or atiter of between about 1 g/liter and about 10 g/liter, about 1.5 g/literand about 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter,about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about4.5 g/liter, or about 1 g/liter and about 4 g/liter. In anotherembodiment, a conditioned cell culture medium according to the inventioncomprises a recombinant glycoprotein at a higher titer than the titerobtained without the use of a medium described herein. In a specificembodiment, the protein or polypeptide is an antibody.

Anti-α4-Integrin Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of particular interest in accordance with the presentinvention. Antibodies are proteins that have the ability to specificallybind a particular antigen. Any anti-α4-integrin antibody that can beexpressed in a host cell can be used in accordance with the presentinvention. In one embodiment, the anti-α4-integrin antibody to beexpressed is a monoclonal antibody.

Particular anti-α4-integrin antibodies can be made, for example, bypreparing and expressing synthetic genes that encode the recited aminoacid sequences or by mutating human germline genes to provide a genethat encodes the recited amino acid sequences. Moreover, theseantibodies can be produced, e.g., using one or more of the followingmethods.

Numerous methods are available for obtaining antibodies, particularlyhuman antibodies. One exemplary method includes screening proteinexpression libraries, e.g., phage or ribosome display libraries. Phagedisplay is described, for example, U.S. Pat. No. 5,223,409; Smith (1985)Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. Thedisplay of Fab's on phage is described, e.g., in U.S. Pat. Nos.5,658,727; 5,667,988; and 5,885,793.

In addition to the use of display libraries, other methods can be usedto obtain an antibody. For example, a protein or a peptide thereof canbe used as an antigen in a non-human animal, e.g., a rodent, i.e., amouse, hamster, or rat.

In one embodiment, the non-human animal includes at least a part of ahuman immunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal antibodies derived from the genes with the desiredspecificity can be produced and selected. See, e.g., XENOMOUSE™, Greenet al. (1994) Nature Genetics 7:13-21, U.S. 2003-0070185, WO 96/34096,and WO 96/33735.

In another embodiment, a monoclonal anti-α4-integrin antibody isobtained from the non-human animal, and then modified, e.g., humanizedor deimmunized. Winter describes an exemplary CDR-grafting method thatcan be used to prepare humanized antibodies described herein (U.S. Pat.No. 5,225,539). All or some of the CDRs of a particular human antibodycan be replaced with at least a portion of a non-human antibody. In oneembodiment, it is only necessary to replace the CDRs required forbinding or binding determinants of such CDRs to arrive at a usefulhumanized antibody that binds to an antigen.

Humanized anti-α4-integrin antibodies can be generated by replacingsequences of the Fv variable region that are not directly involved inantigen binding with equivalent sequences from human Fv variableregions. General methods for generating humanized antibodies areprovided by Morrison, S. L. (1985) Science 229:1202-1207, by Oi et al.(1986) BioTechniques 4:214, and by U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; 5,859,205; and 6,407,213. Those methods include isolating,manipulating, and expressing the nucleic acid sequences that encode allor part of immunoglobulin Fv variable regions from at least one of aheavy or light chain. Sources of such nucleic acid are well known tothose skilled in the art and, for example, can be obtained from ahybridoma producing an antibody against a predetermined target, asdescribed above, from germline immunoglobulin genes, or from syntheticconstructs. The recombinant DNA encoding the humanized antibody can thenbe cloned into an appropriate expression vector. In one embodiment, theexpression vector comprises a polynucleotide encoding a glutaminesynthetase polypeptide. (See, e.g., Porter et al., Biotechnol Prog26(5):1446-54 (2010).). In one embodiment, humanized anti-α4-integrinantibody is natalizumab.

The anti-α4-integrin antibody can include a human Fc region, e.g., awild-type Fc region or an Fc region that includes one or morealterations. In one embodiment, the constant region is altered, e.g.,mutated, to modify the properties of the antibody (e.g., to increase ordecrease one or more of: Fc receptor binding, antibody glycosylation,the number of cysteine residues, effector cell function, or complementfunction). For example, the human IgG1 constant region can be mutated atone or more residues, e.g., one or more of residues 234 and 237.Antibodies can have mutations in the CH2 region of the heavy chain thatreduce or alter effector function, e.g., Fc receptor binding andcomplement activation. For example, antibodies can have mutations suchas those described in U.S. Pat. Nos. 5,624,821 and 5,648,260. Antibodiescan also have mutations that stabilize the disulfide bond between thetwo heavy chains of an immunoglobulin, such as mutations in the hingeregion of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol.Immunol. 30:105-08). See also, e.g., U.S. 2005-0037000.

In other embodiments, the anti-α4-integrin antibody can be modified tohave an altered glycosylation pattern (i.e., altered from the originalor native glycosylation pattern). As used in this context, “altered”means having one or more carbohydrate moieties deleted, and/or havingone or more glycosylation sites added to the original antibody. Additionof glycosylation sites to the presently disclosed antibodies can beaccomplished by altering the amino acid sequence to containglycosylation site consensus sequences; such techniques are well knownin the art. Another means of increasing the number of carbohydratemoieties on the antibodies is by chemical or enzymatic coupling ofglycosides to the amino acid residues of the antibody. These methods aredescribed in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit.Rev. Biochem. 22:259-306. Removal of any carbohydrate moieties presenton the antibodies can be accomplished chemically or enzymatically asdescribed in the art (Hakimuddin et al. (1987) Arch. Biochem. Biophys.259:52; Edge et al. (1981) Anal. Biochem. 118:131; and Thotakura et al.(1987) Meth. Enzymol. 138:350). See, e.g., U.S. Pat. No. 5,869,046 for amodification that increases in vivo half-life by providing a salvagereceptor binding epitope.

The anti-α4-integrin antibodies can be in the form of full lengthantibodies, or in the form of fragments of antibodies, e.g., Fab,F(ab′)₂, Fd, dAb, and scFv fragments. Additional forms include a proteinthat includes a single variable domain, e.g., a camel or camelizeddomain. See, e.g., U.S. 2005-0079574 and Davies et al. (1996) ProteinEng. 9(6):531-7.

In one embodiment, the anti-α4-integrin antibody is an antigen-bindingfragment of a full length antibody, e.g., a Fab, F(ab′)2, Fv or a singlechain Fv fragment. Typically, the anti-α4-integrin antibody is a fulllength antibody. The anti-α4-integrin antibody can be a monoclonalantibody or a mono-specific antibody.

In another embodiment, the anti-α4-integrin antibody can be a human,humanized, CDR-grafted, chimeric, mutated, affinity matured,deimmunized, synthetic or otherwise in vitro-generated antibody, andcombinations thereof.

The heavy and light chains of the anti-α4-integrin antibody can besubstantially full-length. The protein can include at least one, andpreferably two, complete heavy chains, and at least one, and preferablytwo, complete light chains or can include an antigen-binding fragment(e.g., a Fab, F(ab′)2, Fv or a single chain Fv fragment). In yet otherembodiments, the antibody has a heavy chain constant region chosen from,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;particularly, chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, moreparticularly, IgG1 (e.g., human IgG1). Typically, the heavy chainconstant region is human or a modified form of a human constant region.In another embodiment, the antibody has a light chain constant regionchosen from, e.g., kappa or lambda, particularly, kappa (e.g., humankappa).

Interferon Beta-1a Polypeptide

Interferons are a family of naturally-occurring, relatively small,single-chain glycoproteins that are produced by eukaryotic cells inresponse to viral infection and other biological inducers. Interferonsare grouped into three major classes, designated: 1) leukocyteinterferon (interferon-alpha), 2) fibroblast interferon(interferon-beta), and 3) immune interferon (interferon-gamma). Inresponse to viral infection, lymphocytes primarily synthesizeinterferon-alpha (with interferon-gamma), whereas fibroblasts usuallysynthesize interferon-beta. There are two types of Interferon-beta:Interferon beta-1 and Interferon beta-3.

In one embodiment, Interferon beta is a mammalian, human, mouse,humanized or chimeric Interferon beta. In another embodiment, Interferonbeta is a recombinant Interferon beta.

In one embodiment, Interferon beta is Interferon beta-1. In anotherembodiment, Interferon beta-1 is Interferon beta-1a (or Interferonbeta-1a polypeptide). In one embodiment, Interferon beta-1a ismammalian, human, mouse, humanized, or chimeric Interferon beta-1a. Inanother embodiment, Interferon beta-1a is a recombinant Interferonbeta-1a.

In one embodiment, Interferon beta-1a is a fragment of full length,naturally-occurring Interferon beta-1a. In another embodiment,Interferon beta-1a is modified or mutated Interferon beta-1a. In anotherembodiment, Interferon beta-1a is a variant of the naturally-occurringInterferon beta-1a. In one embodiment, Interferon beta-1a is aglycosylation variant.

Interferons with modified activity have been generated (for example,U.S. Pat. Nos. 6,514,729; 4,738,844; 4,738,845; 4,753,795, which areincorporated by reference herein). U.S. Pat. Nos. 5,545,723 and6,127,332, which are incorporated by reference herein in their entirety,disclose mutant interferon beta. Chimeric interferons comprisingsequences from one or more interferons have been made (for example,Chang et al., Nature Biotech. 17(8):793-797 (1999), U.S. Pat. Nos.4,758,428; 5,738,846, which are incorporated by reference in theirentirety). Substitution mutants of interferon beta at positions 49 and51 have also been described (for example, U.S. Pat. No. 6,531,122, whichis incorporated by reference in its entirety). Expression and generationof IFN beta variants (such as glycosylation variants) and conjugateshave been discussed, for example, in U.S. Pat. No. 7,144,574, which isincorporated by reference herein in its entirety.

Interferon-beta variants with enhanced stability have been discussed, inwhich the hydrophobic core was optimized using rational design methods(for example, WO 00/68387, which is incorporated by reference in itsentirety). Alternate formulations that promote interferon stability orsolubility have also been disclosed (for example, U.S. Pat. Nos.4,675,483; 5,730,969; 5,766,582; WO 02/38170, which are incorporated byreference in their entirety).

Interferon-beta mutants with enhanced solubility have been discussed, inwhich several leucine and phenylalanine residues are replaced withserine, threonine, or tyrosine residues (for example, WO 98/48018, whichis incorporated by reference in its entirety). Other modifications toimprove solubility are discussed, for example, in US 2005/0054053 whichis incorporated by reference herein in its entirety. Interferon-betavariants with reduced immunogenicity have been discussed (for example,WO 02/074783, which is incorporated by reference in its entirety).

In one embodiment, the interferon beta-1a polypeptide is AVONEX®.AVONEX® is a 166 amino acid recombinant glycoprotein produced by ChineseHamster Ovary cells and has a molecular weight of approximately 22,500Daltons and amino acid sequence of SEQ ID NO:1.

In one embodiment, the Interferon beta-1a polypeptide is a fragment ofAVONEX®. In another embodiment, Interferon beta-1a is a polypeptidehaving 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with AVONEX®. In oneembodiment, the Interferon beta-1a polypeptide is a variant or mutant ofAVONEX®.

Factor VIII Polypeptide

The Factor VIII polypeptide as used herein is functional factor VIIIpolypeptide in its normal role in coagulation, unless otherwisespecified. Thus, the term Factor VIII includes variant polypeptides thatare functional. Factor VIII proteins can be the human, porcine, canine,and murine factor VIII proteins. The full length polypeptide andpolynucleotide sequences are known, as are many functional fragments,mutants and modified versions. Factor VIII polypeptides include, e.g.,full-length factor VIII, full-length factor VIII minus Met at theN-terminus, mature factor VIII (minus the signal sequence), maturefactor VIII with an additional Met at the N-terminus, and/or factor VIIIwith a full or partial deletion of the B domain. Factor VIII variantsinclude B domain deletions, whether partial or full deletions.

A great many functional factor VIII variants are known, as is discussedabove and below. In addition, hundreds of nonfunctional mutations infactor VIII have been identified in hemophilia patients, and it has beendetermined that the effect of these mutations on factor VIII function isdue more to where they lie within the 3-dimensional structure of factorVIII than on the nature of the substitution (Cutler et al., Hum. Mutat.19:274-8 (2002)), incorporated herein by reference in its entirety. Inaddition, comparisons between factor VIII from humans and other specieshave identified conserved residues that are likely to be required forfunction (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat.No. 6,251,632), incorporated herein by reference in its entirety.

The human factor VIII gene was isolated and expressed in mammalian cells(Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al.,Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337(1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), each of which isincorporated herein by reference in its entirety, and the amino acidsequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199,incorporated herein by reference in its entirety, discloses arecombinant DNA method for producing factor VIII in mammalian host cellsand purification of human factor VIII. Human factor VIII expression inCHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells)has been reported. Human factor VIII has been modified to delete part orall of the B domain (U.S. Pat. Nos. 4,994,371 and 4,868,112, each ofwhich is incorporated herein by reference in its entirety), andreplacement of the human factor VIII B domain with the human factor V Bdomain has been performed (U.S. Pat. No. 5,004,803, incorporated hereinby reference in its entirety). The cDNA sequence encoding human factorVIII and predicted amino acid sequence are shown in SEQ ID NOs:1 and 2,respectively, of US Application Publ. No. 2005/0100990, incorporatedherein by reference in its entirety.

U.S. Pat. No. 5,859,204, Lollar, J. S., incorporated herein by referencein its entirety, reports functional mutants of factor VIII havingreduced antigenicity and reduced immunoreactivity. U.S. Pat. No.6,376,463, Lollar, J. S., incorporated herein by reference in itsentirety, also reports mutants of factor VIII having reducedimmunoreactivity. US Application Publ. No. 2005/0100990, Saenko et al.,incorporated herein by reference in its entirety, reports functionalmutations in the A2 domain of factor VIII.

A number of functional factor VIII molecules, including B-domaindeletions, are disclosed in the following patents U.S. Pat. Nos.6,316,226 and 6,346,513, both assigned to Baxter; U.S. Pat. No.7,041,635 assigned to In2Gen; U.S. Pat. Nos. 5,789,203, 6,060,447,5,595,886, and 6,228,620 assigned to Chiron; U.S. Pat. Nos. 5,972,885and 6,048,720 assigned to Biovitrum, U.S. Pat. Nos. 5,543,502 and5,610,278 assigned to Novo Nordisk; U.S. Pat. No. 5,171,844 assigned toImmuno Ag; U.S. Pat. No. 5,112,950 assigned to Transgene S. A.; U.S.Pat. No. 4,868,112 assigned to Genetics Institute, each of which isincorporated herein by reference in its entirety.

The porcine factor VIII sequence is published, (Toole, J. J., et al.,Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)), incorporated herein byreference in its entirety, and the complete porcine cDNA sequenceobtained from PCR amplification of factor VIII sequences from a pigspleen cDNA library has been reported (Healey, J. F. et al., Blood88:4209-4214 (1996), incorporated herein by reference in its entirety).Hybrid human/porcine factor VIII having substitutions of all domains,all subunits, and specific amino acid sequences were disclosed in U.S.Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093, incorporatedherein by reference in its entirety. More recently, the nucleotide andcorresponding amino acid sequences of the A1 and A2 domains of porcinefactor VIII and a chimeric factor VIII with porcine A1 and/or A2 domainssubstituted for the corresponding human domains were reported in WO94/11503, incorporated herein by reference in its entirety. U.S. Pat.No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA anddeduced amino acid sequences. U.S. Pat. No. 6,458,563, incorporatedherein by reference in its entirety assigned to Emory discloses aB-domain deleted porcine Factor VIII.

In one embodiment, Factor VIII is a recombinant protein comprising humancoagulation Factor VIII (FVIII) covalently linked to the Fc domain ofhuman immunoglobulin G1 (IgG1) (rFVIIIFc). See, e.g., Peters et al., J.of Thrombosis & Haemostasis, 11:132-141 (2012); McCue et al., JChromatogr A., 1216(45):7824-7830 (2009); US 2014/0370035 A1; US2013/0108629, all incorporated herein by reference in their entirety.

In one embodiment, rFVIIIFc was be obtained by transfecting expressionvector into human embryonic kidney 293 cells (HEK293H; Invitrogen) usingLipofectamine 2000 transfection reagent (Invitrogen) at Biogen(Cambridge, Mass.). A stable clonal cell lines were generated byselection with Zeocin (Invitrogen) to produce FVIIIFc. See McCue et al.,J Chromatogr A. 1216(45):7824-7830 (2009)).

Cells

Any eukaryotic cell or cell type susceptible to cell culture can beutilized in accordance with the present invention. For example, plantcells, yeast cells, animal cells, insect cells, avian cells or mammaliancells can be utilized in accordance with the present invention. In oneembodiment, the eukaryotic cells are capable of expressing a recombinantprotein or are capable of producing a recombinant or reassortant virus.

Non-limiting examples of mammalian cells that can be used in accordancewith the present invention include BALB/c mouse myeloma line (NSO/1,ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, TheNetherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells±DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCLS 1); TM cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). In one embodiment, the present invention is used in theculturing of and expression of polypeptides from CHO cell lines. In aspecific embodiment, the CHO cell line is the DG44 CHO cell line. In aspecific embodiment, the CHO cell line is the DUXB11 CHO cell line. In aspecific embodiment, the CHO cell line comprises a vector comprising apolynucleotide encoding a glutamine synthetase polypeptide. In a furtherspecific embodiment, the CHO cell line expresses an exogenous glutaminesynthetase gene. (See, e.g., Porter et al., Biotechnol Prog26(5):1446-54 (2010).). In one embodiment, the present invention is usedin the culturing of and expression of polypeptides from HEK cell lines.

Additionally, any number of commercially and non-commercially availablehybridoma cell lines that express polypeptides or proteins can beutilized in accordance with the present invention. One skilled in theart will appreciate that hybridoma cell lines might have differentnutrition requirements and/or might require different culture conditionsfor optimal growth and polypeptide or protein expression, and will beable to modify conditions as needed.

The eukaryotic cells according to the present invention can be selectedor engineered to produce high levels of protein or polypeptide, or toproduce large quantities of virus. Often, cells are geneticallyengineered to produce high levels of protein, for example byintroduction of a gene encoding the recombinant glycoprotein of interestand/or by introduction of control elements that regulate expression ofthe gene (whether endogenous or introduced) encoding the recombinantglycoprotein of interest.

The eukaryotic cells can also be selected or engineered to survive inculture for extended periods of time. For example, the cells can begenetically engineered to express a polypeptide or polypeptides thatconfer extended survival on the cells. In one embodiment, the eukaryoticcells comprise a transgene encoding the Bcl-2 polypeptide or a variantthereof. See, e.g., U.S. Pat. No. 7,785,880. In a specific embodiment,the cells comprise a polynucleotide encoding the bcl-xL polypeptide.See, e.g., Chiang G G, Sisk W P. 2005. Biotechnology and Bioengineering91(7):779-792.

The eukaryotic cells can also be selected or engineered to modify itsposttranslational modification pathways. In one embodiment, the cellsare selected or engineered to modify a protein glycolsylation pathway.In a specific embodiment, the cells are selected or engineered toexpress an aglycosylated protein, e.g., an aglycosylated recombinantantibody. In another specific embodiment, the cells are selected orengineered to express an afucosylated protein, e.g., an afucosylatedrecombinant antibody.

The eukaryotic cells can also be selected or engineered to allowculturing in serum free medium.

Media

The cell culture of the present invention is prepared in any mediumsuitable for the particular cell being cultured. In some embodiments,the medium contains e.g., inorganic salts, carbohydrates (e.g., sugarssuch as glucose, galactose, maltose or fructose), amino acids, vitamins(e.g., B group vitamins (e.g., B12), vitamin A vitamin E, riboflavin,thiamine and biotin), fatty acids and lipids (e.g., cholesterol andsteroids), proteins and peptides (e.g., albumin, transferrin,fibronectin and fetuin), serum (e.g., compositions comprising albumins,growth factors and growth inhibitors, such as, fetal bovine serum,newborn calf serum and horse serum), trace elements (e.g., zinc,manganese, selenium and tricarboxylic acid intermediates), hydrolysates(hydrolyzed proteins derived from plant or animal sources), andcombinations thereof. Commercially available media such as5×-concentrated DMEM/F12 (Invitrogen), CD OptiCHO feed (Invitrogen), CDEfficientFeed (Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed(Irvine Scientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMDMillipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed BioreactorSupplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria),ActiCHO (PAA/GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ([DMEM], Sigma) are exemplary nutrient solutions. In addition,any of the media described in Ham and Wallace, (1979) Meth. Enz., 58:44;Barnes and Sato, (1980) Anal. Biochem., 102:255; U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; InternationalPublication Nos. WO 90/03430; and WO 87/00195; the disclosures of all ofwhich are incorporated herein by reference, can be used as culturemedia. Any of these media can be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as gentamycin), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range) lipids (such as linoleic or other fatty acids) andtheir suitable carriers, and glucose or an equivalent energy source. Insome embodiments the nutrient media is serum-free media, a protein-freemedia, or a chemically defined media. Any other necessary supplementscan also be included at appropriate concentrations that would be knownto those skilled in the art.

In one embodiment, the mammalian host cell is a CHO cell and a suitablemedium contains a basal medium component such as a DMEM/HAM F-12 basedformulation (for composition of DMEM and HAM F12 media, see culturemedia formulations in American Type Culture Collection Catalogue of CellLines and Hybridomas, Sixth Edition, 1988, pages 346-349) with modifiedconcentrations of some components such as amino acids, salts, sugar, andvitamins, recombinant human insulin, hydrolyzed peptone, such asPrimatone HS or Primatone RL (Sheffield, England), or the equivalent; acell protective agent, such as Pluronic F68 or the equivalent pluronicpolyol; gentamycin; and trace elements. In another embodiment, thesuitable medium contains yeast hydrolysate. In a preferred embodiment,the suitable medium contains yeastolate.

The present invention provides a variety of media formulations that,when used in accordance with other culturing steps described herein,minimize or prevent decreases in cellular viability in the culture whileretaining the ability to control glycosylation of a recombinantglycoprotein of interest.

A media formulation of the present invention that has been shown to beto useful in manipulating glycosylation, while not having greatlynegative impacts on metabolic balance, cell growth and/or viability oron expression of polypeptide or protein comprises the media supplementdescribed herein. One of ordinary skill in the art will understand thatthe media formulations of the present invention encompass both definedand non-defined media.

Cell Culture Processes

Various methods of preparing mammalian cells for production of proteinsor polypeptides by batch and fed-batch culture are well known in theart. A nucleic acid sufficient to achieve expression (typically a vectorcontaining the gene encoding the polypeptide or protein of interest andany operably linked genetic control elements) can be introduced into thehost cell line by any number of well-known techniques. Typically, cellsare screened to determine which of the host cells have actually taken upthe vector and express the polypeptide or protein of interest.Traditional methods of detecting a particular polypeptide or protein ofinterest expressed by mammalian cells include but are not limited toimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, SDS-PAGE, Western blots, enzyme-linkedimmunosorbentassay (ELISA), high performance liquid chromatography(HPLC) techniques, biological activity assays and affinitychromatography. One of ordinary skill in the art will be aware of otherappropriate techniques for detecting expressed polypeptides or proteins.If multiple host cells express the polypeptide or protein of interest,some or all of the listed techniques can be used to determine which ofthe cells expresses that polypeptide or protein at the highest levels.

Once a cell that expresses the polypeptide or protein of interest hasbeen identified, the cell is propagated in culture by any of the varietyof methods well-known to one of ordinary skill in the art. The cellexpressing the polypeptide of interest is typically propagated bygrowing it at a temperature and in a medium that is conducive to thesurvival, growth and viability of the cell. The initial culture volumecan be of any size, but is often smaller than the culture volume of theproduction bioreactor used in the final production of the polypeptide orprotein of interest, and frequently cells are passaged several times inbioreactors of increasing volume prior to seeding the productionbioreactor. The cell culture can be agitated or shaken to increaseoxygenation of the medium and dispersion of nutrients to the cells.Alternatively or additionally, special sparging devices that are wellknown in the art can be used to increase and control oxygenation of theculture. In accordance with the present invention, one of ordinary skillin the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor, including butnot limited to pH, temperature, oxygenation, etc.

The cell density useful in the methods of the present invention can bechosen by one of ordinary skill in the art. In accordance with thepresent invention, the cell density can be as low as a single cell perculture volume. In some embodiments of the present invention, startingcell densities (seed density) can range from about 2×10² viable cellsper mL to about 2×10³, 2×10⁴, 2×10⁵, 2×10⁶, 5×10⁶ or 10×10⁶ viable cellsper mL and higher.

In accordance with the present invention, a cell culture size can be anyvolume that is appropriate for production of polypeptides. In oneembodiment, the volume of the cell culture is at least 500 liters. Inother embodiments, the volume of the production cell culture is 10, 50,100, 250, 1000, 2000, 2500, 5000, 8000, 10,000, 12,000 liters or more,or any volume in between. For example, a cell culture will be 10 to5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 50 to 5,000liters, 50 to 10,000 liters, or 50 to 15,000 liters, 100 to 5,000liters, 100 to 10,000 liters, 100 to 15,000 liters, 500 to 5,000 liters,500 to 10,000 liters, 500 to 15,000 liters, 1,000 to 5,000 liters, 1,000to 10,000 liters, or 1,000 to 15,000 liters. Or a cell culture will bebetween about 500 liters and about 30,000 liters, about 500 liters andabout 20,000 liters, about 500 liters and about 10,000 liters, about 500liters and about 5,000 liters, about 1,000 liters and about 30,000liters, about 2,000 liters and about 30,000 liters, about 3,000 litersand about 30,000 liters, about 5,000 liters and about 30,000 liters, orabout 10,000 liters and about 30,000 liters, or a cell culture will beat least about 500 liters, at least about 1,000 liters, at least about2,000 liters, at least about 3,000 liters, at least about 5,000 liters,at least about 10,000 liters, at least about 15,000 liters, or at leastabout 20,000 liters.

One of ordinary skill in the art will be aware of and will be able tochoose a suitable culture size for use in practicing the presentinvention. The production bioreactor for the culture can be constructedof any material that is conducive to cell growth and viability that doesnot interfere with expression or stability of the produced polypeptideor protein.

The temperature of the cell culture will be selected based primarily onthe range of temperatures at which the cell culture remains viable. Forexample, during the initial growth phase, CHO cells grow well at 37° C.In general, most mammalian cells grow well within a range of about 25°C. to 42° C.

In certain cases, it can be beneficial or necessary to supplement thecell culture during the growth and/or subsequent production phase withnutrients or other medium components that have been depleted ormetabolized by the cells. For example, it might be advantageous tosupplement the cell culture with nutrients or other medium componentsobserved to have been depleted. Alternatively or additionally, it can bebeneficial or necessary to supplement the cell culture prior to thesubsequent production phase. As non-limiting examples, it can bebeneficial or necessary to supplement the cell culture with hormonesand/or other growth factors, particular ions (such as sodium, chloride,calcium, magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, or glucose or otherenergy source.

These supplementary components, including the amino acids, can all beadded to the cell culture at one time, or they can be provided to thecell culture in a series of additions. In one embodiment of the presentinvention, the supplementary components are provided to the cell cultureat multiple times in proportional amounts. In another embodiment, it canbe desirable to provide only certain of the supplementary componentsinitially, and provide the remaining components at a later time. In yetanother embodiment of the present invention, the cell culture is fedcontinually with these supplementary components.

In accordance with the present invention, the total volume added to thecell culture should optimally be kept to a minimal amount. For example,the total volume of the medium or solution containing the supplementarycomponents added to the cell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell cultureprior to providing the supplementary components.

The cell culture can be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc. For example, pH can be controlled bysupplying an appropriate amount of acid or base and oxygenation can becontrolled with sparging devices that are well known in the art.

In certain embodiments of the present invention, the practitioner canfind it beneficial or necessary to periodically monitor particularconditions of the growing cell culture. Monitoring cell cultureconditions allows the practitioner to determine whether the cell cultureis producing recombinant polypeptide or protein at suboptimal levels orwhether the culture is about to enter into a suboptimal productionphase.

In order to monitor certain cell culture conditions, it will benecessary to remove small aliquots of the culture for analysis. One ofordinary skill in the art will understand that such removal canpotentially introduce contamination into the cell culture, and will takeappropriate care to minimize the risk of such contamination.

As non-limiting example, it can be beneficial or necessary to monitortemperature, pH, cell density, cell viability, integrated viable celldensity, lactate levels, ammonium levels, osmolarity, or titer of theexpressed polypeptide or protein. Numerous techniques are well known inthe art that will allow one of ordinary skill in the art to measurethese conditions. For example, cell density can be measured using ahemacytometer, a Coulter counter, or Cell density examination (CEDEX).Viable cell density can be determined by staining a culture sample withTrypan blue. Since only dead cells take up the Trypan blue, viable celldensity can be determined by counting the total number of cells,dividing the number of cells that take up the dye by the total number ofcells, and taking the reciprocal. HPLC can be used to determine thelevels of lactate, ammonium or the expressed polypeptide or protein.Alternatively, the level of the expressed polypeptide or protein can bedetermined by standard molecular biology techniques such as coomassiestaining of SDS-PAGE gels, Western blotting, Bradford assays, Lowryassays, Biuret assays, and UV absorbance. It can also be beneficial ornecessary to monitor the posttranslational modifications of theexpressed polypeptide or protein, including phosphorylation andglycosylation.

The practitioner can also monitor the metabolic status of the cellculture, for example, by monitoring the glucose, lactate, ammonium, andamino acid concentrations in the cell culture, as well as by monitoringthe oxygen production or carbon dioxide production of the cell culture.For example, cell culture conditions can be analyzed by using NOVABioprofile 100 or 400 (NOVA Biomedical, WA). Additionally, thepractitioner can monitor the metabolic state of the cell culture bymonitoring the activity of mitochondria. In one embodiment,mitochondrial activity can be monitored by monitoring the mitochondrialmembrane potential using Rhodamine 123. Johnson L V, Walsh M L, Chen LB. 1980. Proceedings of the National Academy of Sciences 77(2):990-994.

Isolation of Expressed Polypeptide

In general, it will typically be desirable to isolate and/or purifyproteins or polypeptides expressed according to the present invention.In one embodiment, the expressed polypeptide or protein is secreted intothe medium and thus cells and other solids can be removed, as bycentrifugation or filtering for example, as a first step in thepurification process.

Alternatively, the expressed polypeptide can be bound to the surface ofthe host cell. In this embodiment, the media is removed and the hostcells expressing the polypeptide or protein are lysed as a first step inthe purification process. Lysis of mammalian host cells can be achievedby any number of means well known to those of ordinary skill in the art,including physical disruption by glass beads and exposure to high pHconditions.

The polypeptide can be isolated and purified by standard methodsincluding, but not limited to, chromatography (e.g., ion exchange,affinity, size exclusion, and hydroxyapatite chromatography), gelfiltration, centrifugation, or differential solubility, ethanolprecipitation or by any other available technique for the purificationof proteins (See, e.g., Scopes, Protein Purification Principles andPractice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J.and Hames, B. D. (eds.), Protein Expression: A Practical Approach,Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J.N. (eds.), Guide to Protein Purification: Methods in Enzymology (Methodsin Enzymology Series, Vol 182), Academic Press, 1997, all incorporatedherein by reference). For immunoaffinity chromatography in particular,the protein can be isolated by binding it to an affinity columncomprising antibodies that were raised against that protein and wereaffixed to a stationary support. Alternatively, affinity tags such as aninfluenza coat sequence, poly-histidine, or glutathione-S-transferasecan be attached to the protein by standard recombinant techniques toallow for easy purification by passage over the appropriate affinitycolumn. Protease inhibitors such as phenyl methyl sulfonyl fluoride(PMSF), leupeptin, pepstatin or aprotinin can be added at any or allstages in order to reduce or eliminate degradation of the polypeptide orprotein during the purification process. Protease inhibitors areparticularly desired when cells must be lysed in order to isolate andpurify the expressed polypeptide or protein. One of ordinary skill inthe art will appreciate that the exact purification technique will varydepending on the character of the polypeptide or protein to be purified,the character of the cells from which the polypeptide or protein isexpressed, and the composition of the medium in which the cells weregrown.

Pharmaceutical Compositions

A polypeptide can be formulated as a pharmaceutical composition foradministration to a subject, e.g., to treat or prevent a disorder ordisease. Typically, a pharmaceutical composition includes apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Thecomposition can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (See e.g., Berge, S. M., etal. (1977) J. Pharm. Sci. 66:1-19). In one embodiment, a pharmaceuticalcomposition is an immunogenic composition comprising a virus produced inaccordance with methods described herein.

Pharmaceutical formulation is a well-established art, and is furtherdescribed, e.g., in Gennaro (ed.), Remington. The Science and Practiceof Pharmacy, 20^(th) ed., Lippincott, Williams & Wilkins (2000) (ISBN:0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) Ed., Lippincott Williams & Wilkins Publishers (1999)(ISBN: 0683305727); and Kibbe (ed.), Handbook of PharmaceuticalExcipients American Pharmaceutical Association, 3^(rd) ed. (2000) (ISBN:091733096X).

The pharmaceutical compositions can be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The form can depend on the intended mode of administration andtherapeutic application. Typically compositions for the agents describedherein are in the form of injectable or infusible solutions.

In one embodiment, the antibody is formulated with excipient materials,such as sodium chloride, sodium dibasic phosphate heptahydrate, sodiummonobasic phosphate, and a stabilizer. It can be provided, for example,in a buffered solution at a suitable concentration and can be stored at2-8° C.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze drying that yield a powder of an agent describedherein plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the polypeptide can be prepared with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known. See, e.g., Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York(1978).

The foregoing description is to be understood as being representativeonly and is not intended to be limiting. Alternative methods andmaterials for implementing the invention and also additionalapplications will be apparent to one of skill in the art, and areintended to be included within the accompanying claims.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunology4^(th) ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A.Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier HealthSciences Division (2005); Kontermann and Dubel, Antibody Engineering,Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII,Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR PrimerCold Spring Harbor Press (2003).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

EXAMPLES Example 1: Interferon Beta-1a

Interferon beta-1a was produced in a cell culture under controlledconditions using a Chinese Hamster Ovary (CHO) cell line recombinantlyproducing interferon beta-1a. Cells expressing interferon beta-1a wereexpanded from a vial per standard techniques in shake flasks. Toinvestigate the impact of manganese specifically, cells were split fromthe shake flasks into 5 L bioreactors containing medium with variouslevels of manganese supplementation. The only variable in cultivationconditions was the level of manganese supplemented to cell culture andall other medium components and cell culture process conditions wereunchanged. The level of manganese ranged from 0-4800 nM (that is 4.8 μM)supplementation at inoculation on day 0. At harvest, glycosylation,specifically the level of biantennary, TriLac, and triantennary species,of interferon beta-1a was analyzed by LC/MS. Sialylation in the sameexperiment was assessed by a glycan method using 2-aminobenzoic acidlabeling. The data shown in FIG. 1A and FIG. 1B demonstrates that atleast 100 nM manganese, preferably 300 nM manganese, is required inorder to produce consistent glycosylation.

Example 2. rFVIIIFc

rFVIIIFc was produced in a cell culture under controlled conditionsusing a HEK293 cells recombinantly producing rFVIIIFc. Cells expressingrFVIIIFc were expanded from a vial per standard techniques in shakeflasks. To investigate the impact of manganese specifically, cells weresplit from the shake flasks into 5 L bioreactors containing medium withvarious levels of manganese supplementation. The only variable incultivation conditions was the level of manganese supplemented to cellculture and all other medium components and cell culture processconditions were unchanged. The level of manganese ranged from 0-1800 nMmanganese (that is 1.8 μM) supplementation at inoculation on day 0. Atharvest, glycosylation, specifically the level of level of G0+Man6,G1+Man7, and G2+Man9, of rFVIIIFc was analyzed by CELIF. Separatelymannose species were characterized by LC/MS. There was no change inmannosylation (mannose species) level, so the changes in glycosylatedspecies are all changes in galactosylation level. The data shown in FIG.2 demonstrates that at least 50 nM manganese is required to produceconsistent galactosylation.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All documents, articles, publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

1. A method for achieving a predetermined glycosylation profile of ananti-α4-integrin antibody comprising providing manganese to a cellculture at a concentration that falls within a target manganeseconcentration range, wherein the cell culture comprises host cellsproducing the anti-α4-integrin antibody.
 2. The method of claim 1,comprising supplementing the cell culture with manganese if themanganese concentration in the cell culture is below the targetmanganese concentration range.
 3. A method for achieving a predeterminedglycosylation profile of an anti-α4-integrin antibody comprising (i)determining a manganese concentration in a component of a cell culturemedium, (ii) if the manganese concentration is below a target manganeseconcentration range, supplementing the cell culture medium with thecomponent to achieve a manganese concentration within the targetmanganese concentration range, and (iii) culturing a recombinant hostcell producing an anti-α4-integrin antibody in the cell culture mediumcomprising the cell culture medium component.
 4. A method for achievinga predetermined glycosylation profile of an anti-α4-integrin antibodycomprising (i) determining a manganese concentration in a component of acell culture medium, (ii) if the manganese concentration is below atarget manganese concentration range, adding manganese to the componentof the cell culture medium to achieve a manganese concentration withinthe target manganese concentration range, (iii) producing a cell culturemedium using the component of cell culture medium with the targetmanganese concentration, and (iv) culturing a recombinant host cellproducing an anti-α4-integrin antibody in the cell culture mediumcomprising the cell culture medium component with the target manganeseconcentration.
 5. A method for optimizing a cell culture medium for theproduction of an anti-α4-integrin antibody comprising (i) determiningthe amount of manganese in a cell culture medium or a component used toproduce a cell culture medium, and (ii) if the amount of manganese isbelow a target range, supplementing the cell culture medium or thecomponent of the cell culture medium with manganese to achieve an amountof manganese within the target range, wherein the target range issufficient to produce anti-α4-integrin antibody with a predeterminedglycosylation profile.
 6. A method for optimizing a cell culture mediumfor the production of an anti-α4-integrin antibody comprising (i)determining the amount of manganese in a cell culture medium or acomponent used to produce a cell culture medium, and (ii) if the amountof manganese is above a target range, removing manganese from the cellculture medium or the component of the cell culture medium to achieve anamount of manganese within the target range, wherein the target range issufficient to produce anti-α4-integrin antibody with a predeterminedglycosylation profile.
 7. The method of claim 6, wherein thepredetermined glycosylation profile of the anti-α4-integrin antibodycomprises 13 to 32% galactosylation. 8-9. (canceled)
 10. The method ofclaim 6, wherein the predetermined glycosylation profile of theanti-α4-integrin antibody comprises 0.7 to 3.6% sialylation. 11-12.(canceled)
 13. The method of claim 6, wherein the target manganeseconcentration range in the cell culture is 0.025 μM to 10 μM. 14-19.(canceled)
 20. The method of claim 6, wherein the anti-α4-integrinantibody is produced by a eukaryotic host cell. 21-24. (canceled)
 25. Amethod for achieving a predetermined glycosylation profile of aninterferon beta-1a polypeptide comprising providing manganese to a cellculture at a concentration that falls within a target manganeseconcentration range, wherein the cell culture comprises host cellsproducing the interferon beta-1a polypeptide.
 26. The method of claim25, comprising supplementing the cell culture with manganese if themanganese concentration in the cell culture is below the targetmanganese concentration range.
 27. A method for achieving apredetermined glycosylation profile of an interferon beta-1a polypeptidecomprising (i) determining a manganese concentration in a component of acell culture medium, (ii) if the manganese concentration is below atarget manganese concentration range, supplementing the cell culturemedium with the component to achieve a manganese concentration withinthe target manganese concentration range, and (iii) culturing arecombinant host cell producing an interferon beta-1a polypeptide in thecell culture medium comprising the cell culture medium component.
 28. Amethod for achieving a predetermined glycosylation profile of aninterferon beta-1a polypeptide comprising (i) determining a manganeseconcentration in a component of a cell culture medium, (ii) if themanganese concentration is below a target manganese concentration range,adding manganese to the component of the cell culture medium to achievea manganese concentration within the target manganese concentrationrange, (iii) producing a cell culture medium using the component of cellculture medium with the target manganese concentration, and (iv)culturing a recombinant host cell producing an interferon beta-1apolypeptide in the cell culture medium comprising the cell culturemedium component with the target manganese concentration.
 29. A methodfor optimizing a cell culture medium for the production of an interferonbeta-1a polypeptide comprising (i) determining the amount of manganesein a cell culture medium or a component used to produce a cell culturemedium, and (ii) if the amount of manganese is below a target range,supplementing the cell culture medium or the component of the cellculture medium with manganese to achieve an amount of manganese withinthe target range, wherein the target range is sufficient to produceinterferon beta-1a polypeptide with a predetermined glycosylationprofile.
 30. A method for optimizing a cell culture medium for theproduction of an interferon beta-1a polypeptide comprising (i)determining the amount of manganese in a cell culture medium or acomponent used to produce a cell culture medium, and (ii) if the amountof manganese is above a target range, removing manganese from the cellculture medium or the component of the cell culture medium to achieve anamount of manganese within the target range, wherein the target range issufficient to produce interferon beta-1a polypeptide with apredetermined glycosylation profile.
 31. The method of claim 30, whereinthe predetermined glycosylation profile of the interferon beta-1apolypeptide comprises 91 to 100% sialylation. 32-33. (canceled)
 34. Themethod of claim 30, wherein the predetermined glycosylation profile ofthe interferon beta-1a polypeptide comprises 55 to 85% biantennaryglycoproteins. 35-42. (canceled)
 43. The method of claim 30, wherein thetarget manganese concentration range in the cell culture is 0.1 μM to 5μM. 44-48. (canceled)
 49. The method of claim 30, wherein the interferonbeta-1a polypeptide is produced by a eukaryotic host cell. 50-77.(canceled)